Pomegranate supplementation alleviates dyslipidemia and the onset of non-alcoholic fatty liver disease in Wistar rats by shifting microbiota and producing urolithin-like microbial metabolites.

Non-alcoholic fatty liver disease (NAFLD), obesity and related chronic diseases are major non-communicable diseases with high mortality rates worldwide. While dietary sugars are known to be responsible for insulin resistance and metabolic syndrome (MetS), the underlying pathophysiological effects of sustained fructose consumption require further elucidation. We hypothesize that certain bioactive compounds (i.e. punicalagin and ellagic acid) from dietary pomegranate could counteract the harmful effects of sustained fructose consumption in terms of obesity and liver damage. The present study aimed to elucidate both the molecular mechanisms involved in the pathophysiology associated with fructose intake and the effect of a punicalagin-rich commercial pomegranate dietary supplement (P) used as a nutritional strategy to alleviate fructose-induced metabolic impairments. Thus, nineteen Wistar rats fed on a basal commercial feed were supplemented with either 30% (w/v) fructose in drinking water (F; n = 7) or 30% (w/v) fructose solution plus 0.2% (w/v) P (F + P; n = 6) for 10 weeks. The results were compared to those from a control group fed on the basal diet and provided with drinking water (C; n = 6). Body weight and energy intake were registered weekly. P supplementation decreased fat depots, counteracted the dyslipidemia caused by F and improved markers of liver injury including steatosis. The study of the microbiota by metagenomics and urine by untargeted MS-based metabolomics revealed microbial metabolites from P that may be responsible for these health benefits.

Mycobacterium tuberculosis complex in domestic goats in Southern Spain.

Tuberculosis (TB) is a zoonotic infectious disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTC), which can affect a wide variety of domestic and wild animal species. Although the role of goats as a reservoir of MTC bacteria has been evidenced, information about the circulation of MTC strains in this species is still very scarce. The aim of the present study was to determine the seroprevalence, spatial distribution, risk factors and MTC spoligotypes circulating in goats from Andalusia (Southern Spain), the Spanish region with the largest goat census and a hotspot area of TB in both cattle and wild ungulates. A total of 2155 serum samples from 80 goat flocks were analyzed by an in-house ELISA using the P22 protein complex as a coating antigen. Antibodies against MTC were detected in 473 goats (21.9%, 95% CI: 20.2-23.7) and the true seroprevalence was 22.3% (95% CI: 20.6-24.1). Seropositivity was found in 72 (90.0%) of the 80 flocks analyzed. The generalized estimating equation model showed that the management system (higher seroprevalence on intensive and semi-intensive farms), and the presence of hospital pens inside the regular stables, were risk factors potentially associated with MTC exposure in goats in Southern Spain. The spatial analysis identified a significant spatial cluster (p < 0.001) in Eastern Andalusia. A total of 16 different MTC spoligotypes, including five of M. caprae and eleven of M. bovis, were identified in goats between 2015 and 2022 in the study area, with SB0157 as the most frequently isolated. The results obtained indicate widespread and non-homogeneous spatial distribution of MTC in goat herds from Southern Spain. The high individual and herd-level seroprevalence values found suggest that goats could play a significant role in the maintenance and transmission of MTC in the study area. Our results highlight the importance of implementing control measures in this species.

Emergence of epizootic hemorrhagic disease in red deer (Cervus elaphus), Spain, 2022.

Epizootic hemorrhagic disease (EHD) virus serotype 8 (EHDV-8) emerged in Spain in autumn 2022. In this study, we aimed to (1) characterize the clinical and lesional presentation of EHDV infection in European red deer (Cervus elaphus), and (2) study the spatial spread of the virus in wild ruminants in Spain after its introduction, in 2022/2023. We confirmed EHDV infection in two clinically compatible sick red deer by PCR and detection of anti-EHDV specific antibodies. EHDV infection occurred in red deer with hyperacute to acute clinical signs and lesions associated to vascular changes leading to death of the animals. Partial sequences of variable segment 2 (VP2) and segment 5 (NS1) genes of the detected viruses had >99% nucleotide identity with EHDV-8 sequences from Tunisia and Italy. In a cross-sectional serological study of EHDV in 592 wild ruminants, mainly red deer (n=578), in southwestern Spain, we detected anti-EHDV antibodies in 37 of 592 samples (6.3%; 95% confidence interval: 4.3-8.2), all from red deer and from the localities where clinical cases of EHD were confirmed in red deer. We conclude that EHDV-8 infection causes severe EHD in European red deer. The serosurvey revealed a limited spread of EHDV-8 in Spanish wild ruminant populations in the first year of virus detection in Spain.

Fatal leptospirosis in endangered Barbary macaques (Macaca sylvanus) kept in captivity: Assessing the role of sympatric rodents.

Between December 2020 and January 2021, an outbreak of acute mortality in endangered Barbary macaques (Macaca sylvanus) kept in captivity was detected in a zoo in Spain. The main findings observed in the two fatally affected animals at post-mortem evaluation were jaundice, renal tubular necrosis and interstitial nephritis. Leptospira spp. infection was confirmed by real time PCR (qPCR) in different tissues in both individuals. Analyses of secY gene from a positive individual showed 100% homology with a previously published sequence corresponding to Leptospira interrogans serovar Copenhageni. Free-living sympatric brown rats (Rattus norvegicus) from the affected zoo were also analyzed, and showed a prevalence and seroprevalence of Leptospira spp. of 18.2% (4/22; 95% CI: 2.1-34.3) and 41.9% (26/62; 95% CI: 29.7-54.2), respectively. We detected seropositive sera to five different serovars of Leptospira spp. (Copenhageni, Grippotyphosa, Pomona, Canicola and Hardjo) in the rodent population, with L. Copenhageni being the predominant one. This study describes for first time an outbreak of fatal leptospirosis in captive non-human primates in Europe. Our results show that Barbary macaques, an endangered species, are highly susceptible to Leptospira spp. infection, with sympatric wild rodents being the most likely reservoir animals involved in transmission in this outbreak. Our results suggest that rodent control could be an effective measure for minimizing exposure to Leptospira spp. in zoological collections. Given the potential implications for conservation, animal and public health, non-human primates and rodents should be included in surveillance programs for Leptospira spp. in zoos.

Mapping the risk of exposure to Crimean-Congo haemorrhagic fever virus in the Iberian Peninsula using Eurasian wild boar (Sus scrofa) as a model.

Crimean-Congo haemorrhagic fever (CCHF) virus (CCHFV) is a tick-borne zoonotic pathogen that can cause a lethal haemorrhagic disease in humans. Although the virus appears to be endemically established in the Iberian Peninsula, CCHF is an emerging disease in Spain. Clinical signs of CCHFV infection are mainly manifested in humans, but the virus replicates in several animal species. Understanding the determinants of CCHFV exposure risk from animal models is essential to predicting high-risk exposure hotspots for public health action. With this objective in mind, we designed a cross-sectional study of Eurasian wild boar (Sus scrofa) in Spain and Portugal. The study analysed 5,291 sera collected between 2006 and 2022 from 90 wild boar populations with a specific double-antigen ELISA to estimate CCHFV serum prevalence and identify the main determinants of exposure probability. To do so, we statistically modelled exposure risk with host- and environment-related predictors and spatially projected it at a 10 × 10 km square resolution at the scale of the Iberian Peninsula to map foci of infection risk. Fifty-seven (63.3 %) of the 90 populations had at least one seropositive animal, with seroprevalence ranging from 0.0 to 88.2 %. Anti-CCHFV antibodies were found in 1,026 of 5,291 wild boar (19.4 %; 95 % confidence interval: 18.3-20.5 %), with highest exposure rates in southwestern Iberia. The most relevant predictors of virus exposure risk were wild boar abundance, local rainfall regime, shrub cover, winter air temperature and soil temperature variation. The spatial projection of the best-fit model identified high-risk foci as occurring in most of western and southwestern Iberia and identified recently confirmed risk foci in eastern Spain. The results of the study demonstrate that serological surveys of CCHFV vector hosts are a powerful, robust and highly informative tool for public health authorities to take action to prevent human cases of CCHF in enzootic and emergency settings.

Combination with Annual Deworming Treatments Does Not Enhance the Effects of PCV2 Vaccination on the Development of TB in Wild Boar Populations.

Vaccination against PCV2 has been proven to be an effective measure to reduce the severity of TB in wild boar. The combination of this measure with strategies focused on treating other key concomitant pathogens, such as nematodes, could be a useful strategy. This study assesses whether a combination of deworming treatments and PCV2 vaccination may reduce the prevalence and severity of TB in wild boar. The study was conducted on five game estates in mid-western Spain where four groups of wild boar were produced: control, vaccinated, dewormed and vaccinated-dewormed. Wild boars from all groups were hunted between 2017 and 2020, and all of them received a TB diagnosis based on pathological and microbiological tests. Generalised linear models were used to explore the effect of deworming and PCV2 vaccination on TB prevalence and severity. PCV2-vaccinated animals showed lower probabilities of suffering severe TB lesions. However, no differences regarding TB severity were found between dewormed and non-dewormed wild boar. PCV2 vaccination reduces TB severity in wild boar. However, annual deworming does not produce a long-term parasitological reduction that can influence the development of TB in wild boar, nor does it improve the effect of PCV2 vaccination on TB.

Impact of Sustained Fructose Consumption on Gastrointestinal Function and Health in Wistar Rats: Glycoxidative Stress, Impaired Protein Digestion, and Shifted Fecal Microbiota.

The gastrointestinal tract (GIT) is the target of assorted pathological conditions, and dietary components are known to affect its functionality and health. In previous in vitro studies, we observed that reducing sugars induced protein glycoxidation and impaired protein digestibility. To gain further insights into the pathophysiological effects of dietary sugars, Wistar rats were provided with a 30% (w/v) fructose water solution for 10 weeks. Upon slaughter, in vivo protein digestibility was assessed, and the entire GIT (digests and tissues) was analyzed for markers of oxidative stress and untargeted metabolomics. Additionally, the impact of sustained fructose intake on colonic microbiota was also evaluated. High fructose intake for 10 weeks decreased protein digestibility and promoted changes in the physiological digestion of proteins, enhancing intestinal digestion rather than stomach digestion. Moreover, at colonic stages, the oxidative stress was harmfully increased, and both the microbiota and the intraluminal colonic metabolome were modified.

Dirofilaria immitis in pet dogs from the metropolitan area of the Colombian Caribbean.

A cross-sectional study was carried out to determine the frequency and factors associated with Dirofilaria immitis infection in pet dogs in the metropolitan area of the Colombian Caribbean (northern Colombia). A total of 173 dogs were analyzed by a commercial rapid immunochromatographic test (RIT) and a nested PCR of the cytochrome oxidase subunit I gene, in parallel. Ninety-two (53.2%) of the dogs showed positive results to the RIT, while 59 (34.1%) animals had D. immitis DNA by PCR. Positivity to one or both diagnostic techniques was detected in 104 (60.1%; CI95%: 53.8-67.4) of the sampled dogs. In PCR-positive dogs, phylogenetic analyses evidenced high nucleotide identity (100%) with sequences previously obtained from mosquitoes, dogs and other mammals in different countries. Exercise intolerance (p = 0.002; OR: 2.33; CI95%: 1.37-3.96) and thrombocytopenia (p = < 0.001; OR: 1.95; CI95%: 1.11-3.43) were the main factors associated with D. immitis infection in dogs. The high frequency of D. immitis in dogs indicates a wide distribution of this parasite in the metropolitan area of the Colombian Caribbean, which can be of animal and public health concern. Our results highlight the need to combine different methods to increase the diagnostic accuracy of D. immitis.

Monitoring the epidemic of West Nile virus in equids in Spain, 2020-2021.

The largest epidemic of West Nile virus (WNV) reported ever in Spain in both humans and equines occurred in 2020, affecting 77 humans and 139 equine herds. Here, we aimed to monitor the outbreaks detected in equid herds in Andalusia (southern Spain), the Spanish region where 89.9% of the outbreaks were reported, and to evaluate the virus circulation and risk factor associated with WNV exposure in the affected herds. The first WNV case was detected in mid-July 2020, the number of outbreaks peaked in mid-August and the last one was confirmed on 26th October 2020. WNV lineage 1 was detected in 12 clinically affected horses using real time RT-PCR. Molecular analysis evidenced high nucleotide identity with WNV sequences obtained from humans, birds and mosquitoes from Spain and Italy between 2020 and 2022. Between five and eight months after the WNV epidemic, a total of 724 equids (including 485 unvaccinated and 239 vaccinated animals) from 113 of the 125 affected herds in Andalusia were sampled. IgM and IgG antibodies against WNV were detected in 1.6% (8/485; 95%IC: 0.0-2.5) and 61.9% (300/485; 95%IC: 58.3-65.5) of the unvaccinated individuals, respectively. The seropositivity in vaccinated horses was 86.6% (207/239). The main risk factors associated with WNV exposure in unvaccinated equids were the breed (crossbreed), the location of animals in spring-summer (outside), and the presence of natural water ponds close to the surveyed herds. The high individual seroprevalence obtained in the affected herds indicates that WNV circulation was more widespread than the reported by passive surveillance during the WNV epidemic in 2020. The re-emergence of WNV in 2020 in southern Spain evidenced the needed to improve integrated surveillance systems, minimizing the impact of future cases in equids and humans in high-risk areas.

Crosstalk between dietary pomegranate and gut microbiota: evidence of health benefits.

Gut microbiota (GM) is an invisible organ that plays an important role in human health. Increasing evidence suggests that polyphenols in pomegranate (punicalagin, PU) could serve as prebiotics to modulate the composition and function of GM. In turn, GM transform PU into bioactive metabolites such as ellagic acid (EA) and urolithin (Uro). In this review, the interplay between pomegranate and GM is thoroughly described by unveiling a dialog in which both actors seem to affect each other's roles. In a first dialog, the influence of bioactive compounds from pomegranate on GM is described. The second act shows how the GM biotransform pomegranate phenolics into Uro. Finally, the health benefits of Uro and that related molecular mechanism are summarized and discussed. Intake of pomegranate promotes beneficial bacteria in GM (e.g. Lactobacillus spp., Bifidobacterium spp.) while reducing the growth of harmful bacteria (e.g. Bacteroides fragilis group, Clostridia). Akkermansia muciniphila, and Gordonibacter spp., among others, biotransform PU and EA into Uro. Uro contributes to strengthening intestinal barrier and reducing inflammatory processes. Yet, Uro production varies greatly among individuals and depend on GM composition. Uro-producing bacteria and precise metabolic pathways need to be further elucidated therefore contributing to personalized and precision nutrition.

Gut microbiota plays a critical role in maintaining host health.Pomegranate is rich in bioactive components.Consumption of pomegranate positively modulates gut microbiota.Gut microbiota can transform ellagitannins in pomegranate into urolithin.Urolithin has high bioavailability and multiple health benefits.

Effect of feed supplementation with probiotics and postbiotics on strength and health status of honey bee (Apis mellifera) hives during late spring.

Currently, beekeeping faces many risks, such as deteriorating health of honeybees in hives, which results in high mortality rates, mainly during winter. An important consequence is the emergence/re-emergence of communicable diseases such as varroosis or nosemosis. These diseases jeopardize the continuity of the sector because of the absence of effective treatments and harmful residues that they can be retained on wax or honey. This study aimed to evaluate how feed supplementation with probiotic and postbiotic products derived from lactic acid bacteria affected the strength, dynamic population, and sanitary parameters of honey bees. Three groups of 30 hives were established and fed with feed supplemented with control, probiotic, or postbiotic products, with a total of nine applications over two months in late spring. Two monitoring tests were conducted to evaluate the strength and health status of hives. Hives that consumed postbiotic products enhanced their strength, increased bee population and egg laying of the queen, and maintained their reserves of pollen, whereas these parameters decreased in hives belonging to other groups. Furthermore, although the results suggested a favorable effect of postbiotic products on the trend of N. ceranae infection levels, probiotics showed intermediate results. While awaiting long-term results regarding V. destructor infestation, which showed similar trends in all groups, feed supplementation with postbiotics could be an important tool for beekeepers to enhance the strength and health status of their hives.

Melatonin modulates metabolic adaptation of pancreatic stellate cells subjected to hypoxia.

Pancreatic stellate cells (PSCs), the main cell type responsible for the development of fibrosis in pancreatic cancer, proliferate actively under hypoxia. Melatonin has received attention as a potential antifibrotic agent due to its anti-proliferative actions on PSCs. In this work, we investigated the activation of the PI3K/Akt/mTOR pathway and the metabolic adaptations that PSCs undergo under hypoxic conditions, as well as the probable modulation by melatonin. Incubation of cells under hypoxia induced an increase in cell proliferation, and in the expression of alpha-smooth muscle actin and of collagen type 1. In addition, an increase in the phosphorylation of Akt was observed, whereas a decrease in the phosphorylation of PTEN and GSK-3b was noted. The phosphorylation of mTOR and its substrate p70 S6K was decreased under hypoxia. Treatment of PSCs with melatonin under hypoxia diminished cell proliferation, the levels of alpha-smooth muscle actin and of collagen type 1, the phosphorylation of Akt and increased phosphorylation of mTOR. Mitochondrial activity decreased in PSCs under hypoxia. A glycolytic shift was observed. Melatonin further decreased mitochondrial activity. Under hypoxia, no increase in autophagic flux was noted. However, melatonin treatment induced autophagy activation. Nevertheless, inhibition of this process did not induce detectable changes in the viability of cells treated with melatonin. We conclude that PSCs undergo metabolic adaptation under hypoxia that might help them survive and that pharmacological concentrations of melatonin modulate cell responses to hypoxia. Our results contribute to the knowledge of the mechanisms by which melatonin could modulate fibrosis within the pancreas.

Protein carbonylation in food and nutrition: a concise update.

Protein oxidation is a topic of indisputable scientific interest given the impact of oxidized proteins on food quality and safety. Carbonylation is regarded as one of the most notable post-translational modifications in proteins and yet, this reaction and its consequences are poorly understood. From a mechanistic perspective, primary protein carbonyls (i.e. α-aminoadipic and γ-glutamic semialdehydes) have been linked to radical-mediated oxidative stress, but recent studies emphasize the role alternative carbonylation pathways linked to the Maillard reaction. Secondary protein carbonyls are introduced in proteins via covalent linkage of lipid carbonyls (i.e. protein-bound malondialdehyde). The high reactivity of protein carbonyls in foods and other biological systems indicates the intricate chemistry of these species and urges further research to provide insight into these molecular mechanisms and pathways. In particular, protein carbonyls are involved in the formation of aberrant and dysfunctional protein aggregates, undergo further oxidation to yield carboxylic acids of biological relevance and establish interactions with other biomolecules such as oxidizing lipids and phytochemicals. From a methodological perspective, the routine dinitrophenylhydrazine (DNPH) method is criticized not only for the lack of accuracy and consistency but also authors typically perform a poor interpretation of DNPH results, which leads to misleading conclusions. From a practical perspective, the biological relevance of protein carbonyls in the field of food science and nutrition is still a topic of debate. Though the implication of carbonylation on impaired protein functionality and poor protein digestibility is generally recognized, the underlying mechanism of such connections requires further clarification. From a medical perspective, protein carbonyls are highlighted as markers of protein oxidation, oxidative stress and disease. Yet, the specific role of specific protein carbonyls in the onset of particular biological impairments needs further investigations. Recent studies indicates that regardless of the origin (in vivo or dietary) protein carbonyls may act as signalling molecules which activate not only the endogenous antioxidant defences but also implicate the immune system. The present paper concisely reviews the most recent advances in this topic to identify, when applicable, potential fields of interest for future studies.

Host Genetic Diversity and Infectious Diseases. Focus on Wild Boar, Red Deer and Tuberculosis.

Host genetic diversity tends to limit disease spread in nature and buffers populations against epidemics. Genetic diversity in wildlife is expected to receive increasing attention in contexts related to disease transmission and human health. Ungulates such as wild boar (Sus scrofa) and red deer (Cervus elaphus) are important zoonotic hosts that can be precursors to disease emergence and spread in humans. Tuberculosis is a zoonotic disease with relevant consequences and can present high prevalence in wild boar and red deer populations. Here, we review studies on the genetic diversity of ungulates and determine to what extent these studies consider its importance on the spread of disease. This assessment also focused on wild boar, red deer, and tuberculosis. We found a disconnection between studies treating genetic diversity and those dealing with infectious diseases. Contrarily, genetic diversity studies in ungulates are mainly concerned with conservation. Despite the existing disconnection between studies on genetic diversity and studies on disease emergence and spread, the knowledge gathered in each discipline can be applied to the other. The bidirectional applications are illustrated in wild boar and red deer populations from Spain, where TB is an important threat for wildlife, livestock, and humans.

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese.

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.

Melatonin Modulates the Antioxidant Defenses and the Expression of Proinflammatory Mediators in Pancreatic Stellate Cells Subjected to Hypoxia.

Pancreatic stellate cells (PSC) play a major role in the formation of fibrotic tissue in pancreatic tumors. On its side, melatonin is a putative therapeutic agent for pancreatic cancer and inflammation. In this work, the actions of melatonin on PSC subjected to hypoxia were evaluated. Reactive oxygen species (ROS) generation reduced (GSH) and oxidized (GSSG) levels of glutathione, and protein and lipid oxidation were analyzed. The phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and the regulatory protein nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκBα) was studied. The expression of Nrf2-regulated antioxidant enzymes, superoxide dismutase (SOD) enzymes, cyclooxygenase 2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also studied. Total antioxidant capacity (TAC) was assayed. Finally, cell viability was studied. Under hypoxia and in the presence of melatonin generation of ROS was observed. No increases in the oxidation of proteins or lipids were detected. The phosphorylation of Nrf2 and the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1, heme oxygenase-1, SOD1, and of SOD2 were augmented. The TAC was increased. Protein kinase C was involved in the effects of melatonin. Melatonin decreased the GSH/GSSG ratio at the highest concentration tested. Cell viability dropped in the presence of melatonin. Finally, melatonin diminished the phosphorylation of NF-kB and the expression of COX-2, IL-6, and TNF-α. Our results indicate that melatonin, at pharmacological concentrations, modulates the red-ox state, viability, and the expression of proinflammatory mediators in PSC subjected to hypoxia.

Pancreatic stellate cells exhibit adaptation to oxidative stress evoked by hypoxia.

BACKGROUND INFORMATION: Pancreatic stellate cells play a key role in the fibrosis that develops in diseases such as pancreatic cancer. In the growing tumour, a hypoxia condition develops under which cancer cells are able to proliferate. The growth of fibrotic tissue contributes to hypoxia. In this study, the effect of hypoxia (1% O2 ) on pancreatic stellate cells physiology was investigated. Changes in intracellular free-Ca2+ concentration, mitochondrial free-Ca2+ concentration and mitochondrial membrane potential were studied by fluorescence techniques. The status of enzymes responsible for the cellular oxidative state was analyzed by quantitative reverse transcription-polymerase chain reaction, high-performance liquid chromatography, spectrophotometric and fluorimetric methods and by Western blotting analysis. Cell viability and proliferation were studied by crystal violet test, 5-bromo-2-deoxyuridine cell proliferation test and Western blotting analysis. Finally, cell migration was studied employing the wound healing assay. RESULTS: Hypoxia induced an increase in intracellular and mitochondrial free-Ca2+ concentration, whereas mitochondrial membrane potential was decreased. An increase in mitochondrial reactive oxygen species production was observed. Additionally, an increase in the oxidation of proteins and lipids was detected. Moreover, cellular total antioxidant capacity was decreased. Increases in the expression of superoxide dismutase 1 and 2 were observed and superoxide dismutase activity was augmented. Hypoxia evoked a decrease in the oxidized/reduced glutathione ratio. An increase in the phosphorylation of nuclear factor erythroid 2-related factor and in expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 were detected. The expression of cyclin A was decreased, whereas expression of cyclin D and the content of 5-bromo-2-deoxyuridine were increased. This was accompanied by an increase in cell viability. The phosphorylation state of c-Jun NH2 -terminal kinase was increased, whereas that of p44/42 and p38 was decreased. Finally, cells subjected to hypoxia maintained migration ability. CONCLUSIONS AND SIGNIFICANCE: Hypoxia creates pro-oxidant conditions in pancreatic stellate cells to which cells adapt and leads to increased viability and proliferation.

Melatonin modulates red-ox state and decreases viability of rat pancreatic stellate cells.

In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.

Nasal shedding of Mycobacterium tuberculosis in wild boar is related to generalised tuberculosis and concomitant infections.

BACKGROUND: Wild boar is an important reservoir of Mycobacterium tuberculosis variant bovis, the main causative agent of bovine tuberculosis (bTB). A proportion of tuberculosis (TB)-affected wild boars shed M tuberculosis by nasal route, favouring the maintenance of bTB in a multihost scenario. The aim of this work was to assess if M tuberculosis nasal excretion is influenced by factors commonly associated with high TB prevalence in wild boar. METHODS: TB diagnosis and M tuberculosis isolation were carried out in 112 hunted wild boars from mid-western Spain. The association between the presence of M tuberculosis DNA in nasal secretions and explanatory factors was explored using partial least squares regression (PLSR) approaches. RESULTS: DNA from M tuberculosis was detected in 40.8 per cent nasal secretions of the TB-affected animals. Explanatory factors provided a first significant PLSR X's component, explaining 25.70 per cent of the variability observed in M tuberculosis nasal shedding. The presence of M tuberculosis in nasal secretions is more probable in animals suffering from generalised TB and mainly coinfected with Metastrongylus species and porcine circovirus type 2, explaining nearly 90 per cent of the total variance of this model. CONCLUSION: Measures aiming to control these factors could be useful to reduce M tuberculosis shedding in wild boar.

Outbreaks of antimicrobial resistant Salmonella Choleraesuis in wild boars piglets from central-western Spain.

Salmonella enterica serovar Choleraesuis is the aetiological agent of swine paratyphoid being a highly invasive zoonotic pathogen. Wild boar natural populations are experiencing a demographical expansion as well as some farms are breeding this species to release for hunting with management sometimes identical to that of domestic pigs, including supplementation, grouping, and antibiotic treatments. This situation increases the chance of contact between wild boars and livestock, and potentially induces stress, with different sanitary consequences. The present work aims to describe the clinical features of recent outbreaks caused by S. Choleraesuis in wild boar from central-western Spain, as well as the antimicrobial resistance and phylogenetic relationships of isolates involved. 28 strains of S. Choleraesuis were isolated from 28 different wild boars belonging to 10 different game states located in central western Spain and submitted to the Clinical Veterinary Hospital (CVH) of the University of Extremadura. Samples were taken from different organs and cultured according to the ISO 6579:2002 procedure. Suspicious colonies were identified by PCR and antimicrobial resistance was evaluated by disc diffusion susceptibility test and the presence of the main resistance genes as well as 18 plasmid replicons frequently found among the Enterobacteriaceae was verified by PCR. Pulsed field gel electrophoresis was applied to determine the genetic relationship between isolates. The outbreaks under study were characterized by high mortality (35%-84%) and a septicaemic presentation. S. Choleraesuis was isolated from all the wild boars analysed, and 26 of the 28 isolates presented resistance to at least one antibiotic. The predominant resistances found were against sulphonamide, streptomycin, tetracycline, and doxicicline and sul1, strA-strB, and tetA were the most prevalent resistance genes among isolates. 10 strains carried FIIA, FIB+H/1 or FIIA+H/1 plasmids. PFGE classified the isolates into four different profiles, grouped into two clusters. This results show that prevention against S. Choleraesuis must be considered in the sanitary programs of the wild boar breeders.