Enric Vidal - Academia.edu (original) (raw)

Papers by Enric Vidal

PLoS pathogens, Jan 13, 2017

One of the characteristics of prions is their ability to infect some species but not others and p... more One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel trans...

Vaccine, 2016

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control ... more Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.

Procedia in Vaccinology, 2014

Progressin development of tuberculosis (TB) vaccines largely depends on the availability of anima... more Progressin development of tuberculosis (TB) vaccines largely depends on the availability of animal models to test their safety and efficacy before starting with expensive clinical trials. The present study provides a comprehensive evaluation of bacillus Calmette-Guerin (BCG) effects on clinical, immunological, pathological and bacteriological parameters in goats after an experimental challenge with Mycobacterium caprae.Vaccination of goats with BCG reduced the volume of lung gross lesions, the bacterial load in pulmonary lymph nodes and increased the weight gain when compared to unvaccinated animals. Differences in post-challenge IFN-γ responses to ESAT-6/CFP-10 were found to be a useful follow-up biomarker of disease progression and vaccine efficacy. Our results endorse this animal model for further TB vaccine trials.

The Veterinary Journal, 2008

During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an init... more During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an initial reactor was detected using a rapid test on a brain sample. Immunohistochemistry confirmed an atypical TSE presentation that closely resembled the previously described Nor98 cases. Sequencing of the prnp gene confirmed the ARQ/AHQ genotype with the L141F mutation at codon 141 associated with this phenotype. The head, including the brain and cranial lymphoid tissues, was sampled and examined thoroughly. Non-purulent encephalitis, with ectopic lymphoid follicle formation within the brain, was diagnosed concomitant to the TSE. When scrapie-associated prion protein (PrP sc ) deposition was studied by immunohistochemistry there was a noticeable lack of lymphotropism. The distribution of PrP sc in the brain differed considerably from that of classical scrapie cases. Astrogliosis and microgliosis were demonstrated by histochemical procedures.

PLoS ONE, 2013

The ''One world, one health'' initiative emphasizes the need for new strategies to control human ... more The ''One world, one health'' initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirtythree goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (, 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

PLoS ONE, 2012

Elucidation of the structure of PrP Sc continues to be one major challenge in prion research. The... more Elucidation of the structure of PrP Sc continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP Sc ). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometrybased analysis impractical. In order to surmount these difficulties we analyzed PrP Sc from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP Sc samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP Sc . These results reinforce the hypothesis that the structure of PrP Sc consists of a series of highly PK-resistant b-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP Sc is highly resistant to PK and therefore perhaps also contains b-sheet secondary structure. Citation: Vázquez-Fernández E, Alonso J, Pastrana MA, Ramos A, Stitz L, et al. (2012) Structural Organization of Mammalian Prions as Probed by Limited Proteolysis. PLoS ONE 7(11): e50111.

Neurogastroenterology & Motility, 2007

Journal of Wildlife Diseases, 2014

In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead o... more In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead on the Spanish Mediterranean coast. At necropsy, several areas of malacia were macroscopically observed in the periventricular parenchyma of the cerebrum. Microscopically a severe, diffuse, pyogranulomatous, and necrotizing meningoencephalomyelitis was associated with numerous intralesional highly pleomorphic fungal structures. After culture, the fungus, Cunninghamella bertholletiae, was identified by culture and PCR. To our knowledge, this is the first reported case of central nervous system mucormycosis due to Cunninghamella bertholletiae in a cetacean.

Journal of Comparative Pathology, 2003

Disturbances of the g-aminobutyric acid (GABA) neurotransmitter system have been implicated in ch... more Disturbances of the g-aminobutyric acid (GABA) neurotransmitter system have been implicated in chronic degenerative neurological disease. Cognitive dysfunction and neuron loss are features in older dogs. GABAergic neurons also show immunoreactivity for specific calcium-binding proteins. Immunohistochemistry was used to study the neuronal expression of calbindin D-28k and parvalbumin in different areas of the brain in 13 dogs, aged between 2 and 13Á5 years. Calbindin expression was found only in the cerebellum. There were significant differences in the quantity and distribution of neurons expressing these proteins between geriatric and adult brains. Parvalbumin-and calbindin-expressing neurons are relatively sensitive to degeneration in the cerebellum of older dogs. Parvalbumin labelling was associated with dystrophic structures that are commonly associated with ageing.

Journal of Comparative Pathology, 2005

A meningioma with a significant component of granular cells is described in the left ocular orbit... more A meningioma with a significant component of granular cells is described in the left ocular orbit of a 5-year-old male Golden retriever dog that presented with exophthalmos. The neoplastic mass surrounded the optic nerve. Microscopically, the tumour was characterized by nests of large, round to polygonal cells, variable in size, with eccentric nuclei and abundant eosinophilic cytoplasm, containing PAS-positive granules. In focal areas, cells were smaller and occasionally formed whorl-like structures. Immunohistochemical analysis revealed that neoplastic cells reacted positively for vimentin and, with less intensity, for neuron specific enolase and S-100, whereas they were negative for glial fibrillary acidic protein and cytokeratins. Metastatic growths, formed by similar cells, were seen in the lung and heart.

Diseases of Aquatic Organisms, 2011

Dolphin morbillivirus (DMV) has caused 2 epizootics with high mortality rates on the Spanish Medi... more Dolphin morbillivirus (DMV) has caused 2 epizootics with high mortality rates on the Spanish Mediterranean coast, in 1990 and 2006-07, mainly affecting striped dolphins Stenella coeruleoalba. Following the first epizootic unusual DMV infections affecting only the central nervous system of striped dolphins were found, with histological features similar to subacute sclerosing panencephalitis and old dog encephalitis, the chronic latent localised infections caused by defective forms of measles virus and canine distemper virus, respectively. Between 2008 and 2010, monitoring by microscopic and immunohistochemical (IHC) studies of 118 striped dolphins stranded along Catalonia, the Valencia Region and Andalusia showed similar localised DMV nervous system infections in 25.0, 28.6 and 27.4% of cases, respectively, with no significant differences among regions or sex. The body length of DMV-infected dolphins was statistically greater than that of non-infected dolphins (196.5 vs. 160.5 cm; p < 0.001). Molecular detection of DMV was performed by 2 different RT-PCR techniques amplifying a 429 bp fragment and a 78 bp fragment both within the phosphoprotein (P) gene. The 429 bp RT-PCR results contradicted the IHC-DMV results as only 3 of 6 dolphins with positive IHC-DMV had positive PCR results. All 6 cases were positive with the 78 bp RT-PCR. These findings contraindicate the use of the 429 bp RT-PCR protocol based on the P gene to detect this specific form of DMV. DMV localised nervous infection constitutes the most relevant single cause of stranding and death in Mediterranean striped dolphins in the years following a DMV epizootic, and it might even overwhelm the effects of the epizootic itself, at least in 2007.

Vaccine, 2016

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control ... more Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.

Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 2010

In the current study, a rapid chromatographic immunoassay submitted for registration in Europe wa... more In the current study, a rapid chromatographic immunoassay submitted for registration in Europe was used to analyze PrP sc in 13 different areas of brain from 10 confirmed classical scrapie cases. The levels of PrP sc in the different areas of brain were plotted to draw a brain PrP sc distribution curve. This curve was compared with the brain PrP sc distribution curve obtained from immunoblotting and immunohistochemistry tests on the same samples. The distribution of PrP sc in different areas of the brain was similar, irrespective of the test applied, indicating that any of the 3 tests could be used for the characterization of classical cases of scrapie.

ABSTRACT A version of Protein Misfolding Cyclic Amplification (PMCA) based on recombinant PrP (re... more ABSTRACT A version of Protein Misfolding Cyclic Amplification (PMCA) based on recombinant PrP (rec-PrP) as substrate (rec-PMCA) has been used for generating highly resistant PrPres. rec-PrP from hamster, sheep, bank vole, mouse, pig, cow, human, and other species have been seeded with a diversity of prion strains in order to generate self-replicating proteins that display the principal characteristics attributed to mammalian prions. The fact that biochemically and biologically distinguishable rec-PrPSc are being generated in vitro from the same species proves that the GPI and glycosylation components are not necessary in enciphering different strains and different conformations. Electronic microscopy images from all of these rec-PrPres are reminiscent of those observed in GPI-less PrPSc preparations (rods) and clearly are distinguishable from those observed in standard fibril preparations. Infectivity is being tested in different wild type and transgenic mouse models, showing that while the infectivity is high (100% attack rate), new prion strains with atypical characteristics are emerging. rec-PMCA is also being used for estimating the transmission barrier in a more reliable and accurate way. Infectious rec-PrPSc opens new avenues for structural studies. In particular, we have used rec-PMCA to generate enough material to characterize the PK resistant cores of different rPrPSc strains using mass spectrometry. These studies confirm a basic structural similarity of rPrPSc and brain-derived PrPSc.

PLoS pathogens, Jan 13, 2017

One of the characteristics of prions is their ability to infect some species but not others and p... more One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel trans...

Vaccine, 2016

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control ... more Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.

Procedia in Vaccinology, 2014

Progressin development of tuberculosis (TB) vaccines largely depends on the availability of anima... more Progressin development of tuberculosis (TB) vaccines largely depends on the availability of animal models to test their safety and efficacy before starting with expensive clinical trials. The present study provides a comprehensive evaluation of bacillus Calmette-Guerin (BCG) effects on clinical, immunological, pathological and bacteriological parameters in goats after an experimental challenge with Mycobacterium caprae.Vaccination of goats with BCG reduced the volume of lung gross lesions, the bacterial load in pulmonary lymph nodes and increased the weight gain when compared to unvaccinated animals. Differences in post-challenge IFN-γ responses to ESAT-6/CFP-10 were found to be a useful follow-up biomarker of disease progression and vaccine efficacy. Our results endorse this animal model for further TB vaccine trials.

The Veterinary Journal, 2008

During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an init... more During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an initial reactor was detected using a rapid test on a brain sample. Immunohistochemistry confirmed an atypical TSE presentation that closely resembled the previously described Nor98 cases. Sequencing of the prnp gene confirmed the ARQ/AHQ genotype with the L141F mutation at codon 141 associated with this phenotype. The head, including the brain and cranial lymphoid tissues, was sampled and examined thoroughly. Non-purulent encephalitis, with ectopic lymphoid follicle formation within the brain, was diagnosed concomitant to the TSE. When scrapie-associated prion protein (PrP sc ) deposition was studied by immunohistochemistry there was a noticeable lack of lymphotropism. The distribution of PrP sc in the brain differed considerably from that of classical scrapie cases. Astrogliosis and microgliosis were demonstrated by histochemical procedures.

PLoS ONE, 2013

The ''One world, one health'' initiative emphasizes the need for new strategies to control human ... more The ''One world, one health'' initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirtythree goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (, 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

PLoS ONE, 2012

Elucidation of the structure of PrP Sc continues to be one major challenge in prion research. The... more Elucidation of the structure of PrP Sc continues to be one major challenge in prion research. The mechanism of propagation of these infectious agents will not be understood until their structure is solved. Given that high resolution techniques such as NMR or X-ray crystallography cannot be used, a number of lower resolution analytical approaches have been attempted. Thus, limited proteolysis has been successfully used to pinpoint flexible regions within prion multimers (PrP Sc ). However, the presence of covalently attached sugar antennae and glycosylphosphatidylinositol (GPI) moieties makes mass spectrometrybased analysis impractical. In order to surmount these difficulties we analyzed PrP Sc from transgenic mice expressing prion protein (PrP) lacking the GPI membrane anchor. Such animals produce prions that are devoid of the GPI anchor and sugar antennae, and, thereby, permit the detection and location of flexible, proteinase K (PK) susceptible regions by Western blot and mass spectrometry-based analysis. GPI-less PrP Sc samples were digested with PK. PK-resistant peptides were identified, and found to correspond to molecules cleaved at positions 81, 85, 89, 116, 118, 133, 134, 141, 152, 153, 162, 169 and 179. The first 10 peptides (to position 153), match very well with PK cleavage sites we previously identified in wild type PrP Sc . These results reinforce the hypothesis that the structure of PrP Sc consists of a series of highly PK-resistant b-sheet strands connected by short flexible PK-sensitive loops and turns. A sizeable C-terminal stretch of PrP Sc is highly resistant to PK and therefore perhaps also contains b-sheet secondary structure. Citation: Vázquez-Fernández E, Alonso J, Pastrana MA, Ramos A, Stitz L, et al. (2012) Structural Organization of Mammalian Prions as Probed by Limited Proteolysis. PLoS ONE 7(11): e50111.

Neurogastroenterology & Motility, 2007

Journal of Wildlife Diseases, 2014

In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead o... more In May 2012, an adult, male bottlenose dolphin (Tursiops truncatus) was found stranded and dead on the Spanish Mediterranean coast. At necropsy, several areas of malacia were macroscopically observed in the periventricular parenchyma of the cerebrum. Microscopically a severe, diffuse, pyogranulomatous, and necrotizing meningoencephalomyelitis was associated with numerous intralesional highly pleomorphic fungal structures. After culture, the fungus, Cunninghamella bertholletiae, was identified by culture and PCR. To our knowledge, this is the first reported case of central nervous system mucormycosis due to Cunninghamella bertholletiae in a cetacean.

Journal of Comparative Pathology, 2003

Disturbances of the g-aminobutyric acid (GABA) neurotransmitter system have been implicated in ch... more Disturbances of the g-aminobutyric acid (GABA) neurotransmitter system have been implicated in chronic degenerative neurological disease. Cognitive dysfunction and neuron loss are features in older dogs. GABAergic neurons also show immunoreactivity for specific calcium-binding proteins. Immunohistochemistry was used to study the neuronal expression of calbindin D-28k and parvalbumin in different areas of the brain in 13 dogs, aged between 2 and 13Á5 years. Calbindin expression was found only in the cerebellum. There were significant differences in the quantity and distribution of neurons expressing these proteins between geriatric and adult brains. Parvalbumin-and calbindin-expressing neurons are relatively sensitive to degeneration in the cerebellum of older dogs. Parvalbumin labelling was associated with dystrophic structures that are commonly associated with ageing.

Journal of Comparative Pathology, 2005

A meningioma with a significant component of granular cells is described in the left ocular orbit... more A meningioma with a significant component of granular cells is described in the left ocular orbit of a 5-year-old male Golden retriever dog that presented with exophthalmos. The neoplastic mass surrounded the optic nerve. Microscopically, the tumour was characterized by nests of large, round to polygonal cells, variable in size, with eccentric nuclei and abundant eosinophilic cytoplasm, containing PAS-positive granules. In focal areas, cells were smaller and occasionally formed whorl-like structures. Immunohistochemical analysis revealed that neoplastic cells reacted positively for vimentin and, with less intensity, for neuron specific enolase and S-100, whereas they were negative for glial fibrillary acidic protein and cytokeratins. Metastatic growths, formed by similar cells, were seen in the lung and heart.

Diseases of Aquatic Organisms, 2011

Dolphin morbillivirus (DMV) has caused 2 epizootics with high mortality rates on the Spanish Medi... more Dolphin morbillivirus (DMV) has caused 2 epizootics with high mortality rates on the Spanish Mediterranean coast, in 1990 and 2006-07, mainly affecting striped dolphins Stenella coeruleoalba. Following the first epizootic unusual DMV infections affecting only the central nervous system of striped dolphins were found, with histological features similar to subacute sclerosing panencephalitis and old dog encephalitis, the chronic latent localised infections caused by defective forms of measles virus and canine distemper virus, respectively. Between 2008 and 2010, monitoring by microscopic and immunohistochemical (IHC) studies of 118 striped dolphins stranded along Catalonia, the Valencia Region and Andalusia showed similar localised DMV nervous system infections in 25.0, 28.6 and 27.4% of cases, respectively, with no significant differences among regions or sex. The body length of DMV-infected dolphins was statistically greater than that of non-infected dolphins (196.5 vs. 160.5 cm; p < 0.001). Molecular detection of DMV was performed by 2 different RT-PCR techniques amplifying a 429 bp fragment and a 78 bp fragment both within the phosphoprotein (P) gene. The 429 bp RT-PCR results contradicted the IHC-DMV results as only 3 of 6 dolphins with positive IHC-DMV had positive PCR results. All 6 cases were positive with the 78 bp RT-PCR. These findings contraindicate the use of the 429 bp RT-PCR protocol based on the P gene to detect this specific form of DMV. DMV localised nervous infection constitutes the most relevant single cause of stranding and death in Mediterranean striped dolphins in the years following a DMV epizootic, and it might even overwhelm the effects of the epizootic itself, at least in 2007.

Vaccine, 2016

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control ... more Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.

Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc, 2010

In the current study, a rapid chromatographic immunoassay submitted for registration in Europe wa... more In the current study, a rapid chromatographic immunoassay submitted for registration in Europe was used to analyze PrP sc in 13 different areas of brain from 10 confirmed classical scrapie cases. The levels of PrP sc in the different areas of brain were plotted to draw a brain PrP sc distribution curve. This curve was compared with the brain PrP sc distribution curve obtained from immunoblotting and immunohistochemistry tests on the same samples. The distribution of PrP sc in different areas of the brain was similar, irrespective of the test applied, indicating that any of the 3 tests could be used for the characterization of classical cases of scrapie.

ABSTRACT A version of Protein Misfolding Cyclic Amplification (PMCA) based on recombinant PrP (re... more ABSTRACT A version of Protein Misfolding Cyclic Amplification (PMCA) based on recombinant PrP (rec-PrP) as substrate (rec-PMCA) has been used for generating highly resistant PrPres. rec-PrP from hamster, sheep, bank vole, mouse, pig, cow, human, and other species have been seeded with a diversity of prion strains in order to generate self-replicating proteins that display the principal characteristics attributed to mammalian prions. The fact that biochemically and biologically distinguishable rec-PrPSc are being generated in vitro from the same species proves that the GPI and glycosylation components are not necessary in enciphering different strains and different conformations. Electronic microscopy images from all of these rec-PrPres are reminiscent of those observed in GPI-less PrPSc preparations (rods) and clearly are distinguishable from those observed in standard fibril preparations. Infectivity is being tested in different wild type and transgenic mouse models, showing that while the infectivity is high (100% attack rate), new prion strains with atypical characteristics are emerging. rec-PMCA is also being used for estimating the transmission barrier in a more reliable and accurate way. Infectious rec-PrPSc opens new avenues for structural studies. In particular, we have used rec-PMCA to generate enough material to characterize the PK resistant cores of different rPrPSc strains using mass spectrometry. These studies confirm a basic structural similarity of rPrPSc and brain-derived PrPSc.