Pollinator declines threaten biodiversity and food security, with honey bees (Apis mellifera) suffering widespread losses due to multiple interacting stressors. Pathogens -including Varroa destructor, associated viruses, and Vairimorpha (Nosema) ceranae- form complex antagonistic communities within honey bee colonies, yet the structure of these interactions remains poorly understood. Evaluating spatial and temporal variation in hostpathogen networks is essential for predicting colony performance. This study aimed to (1) characterize the individual-based network of honey bee colonies and their pathogens, and (2) assess how network structure relates to colony population dynamics and productivity. To do so we sampled similar to 400 honey bee colonies in the region of Extremadura (SW Spain), encompassing both spatial and temporal variability at smaller (apiary, season) and larger (locality, year) scales. We measured variables related to population dynamics (adult bees, brood) and colony productivity (pollen, honey), as well as loads of four of the most important pathogens of honey bees, V. destructor, N. ceranae, DWV and CBPV, in order to relate interactions with pathogens with colony performance. Host-pathogen interactions showed strong spatial and temporal structuring, with colonies dominated by a single pathogen in specific locations and in specific times. This generated distinct network modules differing in infection levels, population dynamics, and productivity. Colony declines were mainly driven by overall pathogen load and V. destructor interaction frequency, although some low-performing modules were unrelated to pathogen infection, suggesting additional environmental stressors. These findings indicate that 1) targeted management of the most vulnerable network modules could be more efficient than broad sanitation strategies, and 2) network modularity may prevent pathogen spread, so avoiding connecting modules may ease management strategies.

The isolation of non-tuberculous mycobacteria (NTM) is common in cattle positive for tuberculosis (TB) in official diagnostic tests, whereas data on specific NTM species in Spanish cattle remain limited. This study identifies the most frequently isolated NTM species from Single Intradermal Tuberculin Test (SITT)-positive cattle in Extremadura, western Spain. Among 1669 Mycobacterium Growth Indicator Tube (MGIT) positive cultures collected in 2018, 493 (29.54 %) were identified as NTM, and 194 were randomly selected for further analysis. Polymerase Chain Reaction (PCR)-restriction analysis of the hsp65 gene and partial sequencing of 16S ribosomal DNA (rDNA) confirmed a diverse range of species. The most prevalent complex was Mycobacterium avium (40.12 %), including M. senriense, M. intracellulare, and M. avium subsp. paratuberculosis. Other notable NTM species (23.35 %) included M. bourgelatii, M. kansasii, M. gordonae, and M. shinjukuense. Less frequent complexes included M. simiae (11.38 %), M. ulcerans (3.59 %), M. parafortuitum (2.99 %), and M. terrae (1.20 %), along with M. holsaticum (1.20 %), a species related to the M. tuberculosis complex. Phylogenetic analysis and geographic mapping revealed weak correlation between genetic and geographic distances (Mantel test: Rxy = 0.015, P = 0.253), suggesting limited spatial structuration of genetic diversity. Alpha diversity metrics indicated moderate diversity (Shannon's H = 2.641, Simpson's D = 0.106), with some zones exhibiting greater species evenness. Diversity analyses showed moderate dissimilarity among clusters. These findings enhance understanding of Mycobacterium diversity and distribution while emphasizing the diagnostic challenges posed by NTM in TB detection and the importance of molecular tools in species identification and epidemiological surveillance.

PCV-3 and PCV-4 are novel viruses that can infect domestic pigs and wild boars. Both viruses are associated with multiple disorders in domestic pigs (reproductive failure, respiratory damage, etc.). However, the clinical impact on wild boars remains unknown. This study aimed to assess the presence of these viruses in wild boars from mid-western Spain and their sanitary impact on the species. A total of 166 submandibular lymph nodes were collected from hunted wild boars, along with available information about their reproductive status, lung injuries, body condition, and tuberculosis status. The samples were used to detect PCV-3 and PCV-4 using real-time PCR. In total, 84.9% of the sampled animals were positive for PCV-3, and 33.7% were positive for PCV-4. The detection of PCV-4 was more frequent in wild boars that had received supplementary feeding, suggesting that the direct contact favored by this practice could increase the transmission of this virus in wild boar populations. The infections did not seem to influence the body condition, reproductive status, lung lesions, or TB lesion severity patterns in the studied animals. Thus, although these viruses have been widely detected throughout wild boar populations from the studied area, they do not seem to be a health threat to this species. Nevertheless, their monitoring in wild boars is recommended, as they are often in contact with extensively reared pigs, which are susceptible to these viruses.

Varroa destructor is a parasitic mite of honeybees that is considered one of the main problems in beekeeping today. The reduction in the efficacy of conventional miticides and the presence of residues in beehives highlight the need to investigate new organic products as alternative treatments. Postbiotic products have been shown to decrease mite viability in in vitro experiments. However, their use in beehives has not yet been assessed. The aim of this study was to test the efficacy of postbiotics and oxalic acid against V. destructor under field conditions. Two experiments were performed during the summer and autumn seasons, with four study groups: control (C), postbiotic (POS), oxalic acid (OX), and both combined (POX). Phoretic and brood V. destructor were determined at the beginning and end of the study to assess their evolution, as well as the percentage effectiveness of each treatment by registering the mite fall in beehive sanitary bottoms. Postbiotic alone did not show a significant effect on V. destructor under field conditions. However, the combined treatment of postbiotics and oxalic acid improved the results obtained with oxalic acid alone, resulting in greater effectiveness and reduction of phoretic and brood V. destructor.

Feed supplementation in beehives is a commonly necessary practice in beekeeping today, to address the many risks the honey bees face, mainly the climate change and times of food scarcity, agricultural pesticides, or pathogens such as Varroa destructor or Nosema ceranae . Protein supplements and postbiotics products have been evaluated in beehives feed, obtaining improvements of the strength and sanitary status, but they have not been tested in combination. In this study, two experiences have been carried out in autumn and spring to check the potential beneficial effect of this combination in the beehives. Two monitoring were carried out, before and after 1 month of the application of products, in order to assess the strength (number of bees, amount of brood, brood survival, and pollen/honey reserves) and sanitary status ( V. destructor and N. ceranae infestation levels) of the colonies. Firstly, the results obtained in autumn experience showed a reduction of both pathogens in supplemented group. On the other hand, in spring, the combination of protein and postbiotics enhanced the brood survival and honey yield, and decreased the V. destructor infestation. Thus, the combined use of protein and postbiotics as feed supplements could be an important tool to improve the sanitary status after the cold season, and to increase the strength and production of beehives in spring, unifying the benefits of both supplements.

Nutrition is key to improve honey bees' resilience to environmental stress factors that threaten their health. Yet, little is known about the micronutritional needs of colonies, in particular about molybdenum, an essential trace element in biology. This study focuses on a coordination complex based on molybdenum Mo(+V) which was assessed for compatibility with standard beekeeping practices. It was found to be non-toxic for bees, stable and easy to use. Importantly, it does not leave any residues in honey produced. This study investigated how supplementation with few milligrams of this complex can improve hive performance at early spring and reduce winter mortality. Over a two-year field campaign, 283 beehives spread over 6 apiaries located in Spain, Greece and France were involved: 142 beehives supplemented with the molybdenum-based compound and 141 beehives as a control group. Supplementation resulted in a significant reduction in winter colony losses, averaging a 44% decrease, ranging between 27% and 75% in the different apiaries. The risk of death was twice lower in the Na-Mo2O4-EDTA group compared to the control group (risk ratio: 0.51). The impact on bee populations and honey production was also evaluated. A significant increase in honey reserves within the brood area of 107% was observed in the Greek apiary, whereas no comparable effect was detected in Spain, suggesting that local environmental conditions or management practices may influence this parameter. This study highlights the importance of molybdenum in the management of honey bees as an efficient tool to reduce the winter mortality of the colonies.

The pesticide residues in beeswax pose a risk to bee health and hive productivity. This study analyzed 25 beeswax samples from apiaries across five regions in Extremadura, a key beekeeping area in Spain. Foundation wax samples were also sourced from three common suppliers to evaluate contamination at the origin. A multi-residue of 560 substances was performed using QuEChERS extraction, LC-MS, and GC-MS methods. The results revealed 18 insecticides, 7 fungicides, and 4 herbicides, including banned substances such as endrin (up to 218.57 µg/kg) and chlorfenvinphos (15-16 µg/kg). Acrinathrin (5.27-122 µg/kg) and tau-fluvalinate (6.5-138.79 µg/kg) were the most frequent residues. Foundation wax accumulated 16 pesticide residues, highlighting its potential as a source of contamination. Banned pesticides have also been detected. These findings highlight the importance of monitoring pesticide residues in commercial foundation waxes and assessing the impact of pesticide exposure from wax on beekeeping and human safety.

Porcine circovirus 4 (PCV4) was first identified in China in 2019 and retrospectively traced back to 2008. However, unlike other circoviruses, its distribution appeared to be largely confined to Asian countries until recent reports from Spain and the United States. This study aims to enhance knowledge of the past and present circulation of PCV4 in non-Asian countries, particularly in Spain and in wild boars, where it was previously detected at a significant prevalence. By genetically characterizing the strains, the contextualization within both national and international epidemiological frameworks was attempted. A total of 302 lymph node samples were tested, with 62 testing positives by quantitative real-time polymerase chain reaction (qPCR), predominantly from the 2011 to 2015 period, although five positives were detected in the 2022-2024. Complete open reading frame 2 (ORF2) sequences were obtained from 10 strains. Phylogenetic analysis revealed two major clusters: one comprising only Chinese sequences and another containing strains from multiple continents, including Spain and the United States. The Spanish strains formed a distinct monophyletic clade whose introduction in the country was estimated through phylodynamic analyses around 2000, suggesting long-term undetected circulation. Within Spain, a progressive geographical spread and strain exchange between wild boars and outdoor reared domestic pigs could be inferred. On the other hand, the lack of detection in intensively raised pigs, at least in Europe, remains unclear. The present findings extend the current knowledge of PCV4 history outside Asia and challenge the paradigm of a recent international spreading. Recognizing the current uncertainties in PCV4's international distribution and epidemiology, further efforts are needed, including the use of archived samples in diagnostic activities as well as the sharing of negative results.

Animal tuberculosis (TB) and porcine circovirus 2 (PCV-2) coinfections have been associated to the development of severe patterns of tuberculous lesions. These coinfections are frequently observed in wild boar, considered the main wild reservoir of TB in Spain. The vaccination of wild boar with a single dose of PCV-2 vaccine has been associated with less severity of lesions. However, the underlying immune mechanisms affecting these animals remain scarcely known. The aim of this study was to determine whether PCV-2 vaccination alters cytokine concentrations in the serum of wild boars naturally coinfected with TB and PCV-2. Serum samples were collected from hunted wild boar in game estates where part of the population had been previously vaccinated against PCV-2. Serum cytokine concentrations were measured using the MILLIPLEX MAP multiplex assay, and the results were analysed using Principal Component Analysis (PCA). The results suggest significant differences in the concentrations of IL-1β, IL-2, IL-10, IL-12, and IL-18 cytokines between PCV-2-vaccinated and non-vaccinated animals, with lower levels observed in the vaccinated group. IL-1β, IL-2, IL-12 and IL-18 are pro-inflammatory cytokines involved in Th1 response. Exacerbated inflammatory responses can result in more severe lesional patterns. Therefore, the reduced levels of these cytokines observed in PCV-2-vaccinated animals could be associated with the presence of less severe tuberculous lesional patterns.

The Iberian lynx (Lynx pardinus) population has been increasing in recent years thanks to the collaboration of several associations within the framework of the Life Lynxconnect project, which promotes captive breeding for the subsequent release of specimens into the environment. It is therefore important to know their population status, the diseases to which these animals are exposed, and how they affect their repopulation. In this sense, this study aims to study how the presence of mycobacteria affects the lynx population in Extremadura (southwest of Spain). To this end, blood samples, tracheal swabs, and tracheobronchial washings from live-captured lynxes, as well as mediastinal and mesenteric lymph nodes from roadkill animals, were collected. The association between the presence of mycobacteria and various factors—including body condition, age, sex, and blood parameters—was subsequently evaluated. Up to date, our findings show the absence of tuberculosis (TB) in all lynxes tested, while they seem to be reservoirs of non-tuberculous mycobacteria (NTM), mainly M. lentiflavum, the most isolated species. Nonetheless, these mycobacteria appear to have no significant effect on the health condition of the animals.

The Eurasian wild boar (Sus scrofa) is a key wildlife host for tuberculosis (TB) in central and southwestern Spain, posing a challenge to TB eradication in livestock. New strategies, including the use of beneficial microbes, are being explored to mitigate wildlife diseases. This study evaluated the effect of oral supplementation with postbiotic antimycobacterial metabolites produced using Ingulados’ lactic acid bacteria (LAB) collection on TB development in wild boar. A total of 20 game estates in mid-western Spain were divided into two groups: one fed with standard feed containing the postbiotic product and a control group fed without postbiotics. Data were collected from wild boar during hunting events pre- and post-supplementation. The presence of TB-like lesions (TBLLs), lesion severity and seropositivity against Mycobacterium bovis were assessed. Postbiotic supplementation led to a 36.87% reduction in TBLLs and a 35.94% decrease in seropositivity. Notably, young wild boar showed a 64.72% reduction in TBLLs and an 81.80% drop in seropositivity, suggesting reduced transmission. These findings support the potential of postbiotics as a safe, feasible and sustainable tool to control TB in wild boar, offering a promising addition to broader TB eradication efforts.

Wild boar (Sus scrofa) is a common wild ungulate known as the most important reservoir of tuberculosis (TB) in Spain. The severity of TB lesions in this species and the high prevalence of porcine circovirus type 2 (PCV-2) have been related. PCV-2 is ubiquitous in swine populations, being usual for the free-living ones the contact with this agent. Recent studies found a correlation between a decrease of generalised TB prevalence in wild boar populations and the PCV-2-vaccination. The aim of this study was to find out if PCV-2 vaccination modulates the gene expression of cytokines from immune cells after its exposition with mycobacterial antigens using an in vitro methodology. A total of 46 wild boars from a PCV-2 infection endemic area were blood-sampled before and after the PCV-2 vaccination of 22 of them. Peripheral blood mononuclear cells (PBMC) were obtained and isolated from these samples. Aliquots of the cells were in vitro cultured and respectively stimulated with PPDa, PPDb, and a mitogen. A complete analysis of the gene expression of cytokines from the cultured PBMC was carried out. Also, Mycobacterium bovis and PCV-2 contacts were revealed by ELISA and/or qPCR. The results demonstrated that the animals which have had contact with PCV-2 and had been vaccinated, manifested a significant decrease in gene expression of proinflammatory cytokines, like interleukin 1 beta, interleukin 6, and tumour necrosis factor-alpha, possibly related with the severity of TB lesions, and also a significant decrease of interleukin 10, a key cytokine. In conclusion, in case of possible infection or contact events with the virus, PCV-2 vaccination could be an effective measure to reduce the TB severity in wild boar populations, which could decrease the intra and interspecies transmission of TB.

Honey bees assume a pivotal role as primary pollinators, but they are currently facing a growing crisis of colony losses on a global scale. This sector is important for generating essential products, preserving ecosystems, and crop pollination. This study includes the sampling of 179 beehives from three apiaries in the traditional beekeeping area of Extremadura (Spain) vital beekeeping sector and was carried out between 2020 and 2021 using the decision trees-based model. Some studies have tried to identify the primary causative factors of this issue. However, it is insufficient because the approach disregards potential nonlinear interactions among the various factors. For this reason, through meticulous exploration of different causative factors including Varroa destructor, Nosema ceranae, Deformed Wing Virus (DWV), Chronic Bee Paralysis Virus (CBPV), and strength factors, our study employed for first time machine learning methods to identify the most important variables generating colony loss. Our analysis underscores the importance of brood levels (operculated and open), pollen and honey, Varroa destructor infestation, virus (DWV), and honey bee populations as key determinants of colony survival. These findings hold promise for guiding efficacious colony management strategies and underscoring the latent potential of machine-learning applications in the realm of beekeeping.

Colony collapse disorder (CCD) has affected bees worldwide in recent decades, with southwestern Spain being no exception. This disorder is one of the main causes of Apis mellifera mortality and is believed to be caused by environmental, social and sanitary conditions. Dietary supplementation can help to improve some parameters of the general status and sanitary condition of bees, such as infestation by certain recurrent pathogens, including Varroa destructor and Nosema ceranae, by enhancing immune and social response. Thus, the aim of this study was to test a liquid hydrolysed protein supplement on the health and general status of the hive in several apiaries with access to the same natural food and under similar climatic conditions. We selected two groups of ten hives (supplemented by either placebo or protein) from five apiaries where the number of adult bees, amount of brood (open and operculated), honey and pollen reserves, infestation by V. destructor, N. ceranae, deformed wing virus (DWV) and chronic bee paralysis virus (CBPV) were measured. Additionally, we assess the expression of four immune system-related genes and a gene encoding vitellogenin. At the end of this work, treated hives showed a significant increase in open brood and a decrease in V. destructor infestation. Also, these hives showed a significant decrease in the mortality rate after the cold season. Therefore, supplementation with this product improved the health of the hive and could be a promising tool against bee colony loss.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host–pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-β, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

In hunted animals, quality of blood samples may often be compromised. Alternative samples, such as meat juice, may offer an advantage to perform serological tests. This study evaluates if meat juice is a feasible alternative sample to perform the Tuberculosis ELISA test in hunted large game. Between 2017 and 2022, 175 samples were collected from 97 animals (14 red deer + 83 wild boar) in Portugal and Spain. Cohen's kappa coefficient was calculated at 0.71, pointing out a good agreement using 156 paired samples. The sensitivity of the ELISA test with serum was 37.6%, considering Tuberculosis-like lesions (TBL) detected during the initial examination (26 TBL+/ELISA+ in a total of 78 serum samples). Using meat juice as matrix, the sensitivity increased to 37.5% (33 TBL+/ELISA+ in 97 meat juice samples). According to the agreement score and sensitivity being so close between the two matrices tested, meat juice could be a feasible alternative matrix.

The Varroa destructor mite infests Apis mellifera colonies and causes significant harm. Traditional treatments have become less effective because of mite resistance development and can also generate residues inside beehives. This study aimed to gauge the efficacy of a beehive-derived postbiotic in reducing V. destructor viability and to explore its synergies with organic compounds. Four lactic acid bacteria (LAB) species, Leuconostoc mesenteroides, Lactobacillus helsingborgensis, Bacillus velezensis, and Apilactobacillus kunkeei, were isolated and tested in a postbiotic form (preparations of inanimate microorganisms and/or their components) via bioassays. L. mesenteroides, L. helsingborgensis, and B. velezensis notably reduced the mite viability compared to the control, and they were further tested together as a single postbiotic product (POS). Further bioassays were performed to assess the impact of the POS and its combinations with oxalic acid and oregano essential oil. The simple products and combinations (POS/Oregano, POS/Oxalic, Oregano/Oxalic, and POS/Oregano/Oxalic) decreased the mite viability. The most effective were the oxalic acid combinations (POS/Oregano/Oxalic, Oxalic/Oregano, POS/Oxalic), showing significant improvements compared to the individual products. These findings highlight the potential of combining organic products as a vital strategy for controlling V. destructor infection. This study suggests that these combinations could serve as essential tools for combating the impact of mites on bee colonies.

Rabbits (Oryctolagus cuniculus) are vitally important species in the Iberian Peninsula ecosystem. However, since 1950, there has been a significant population decline, with major repercussions. This situation is mainly due to the presence of infectious diseases, such as myxomatosis, which is expanding and is characterized by severe and fatal clinical manifestations. Current control measures, mainly those based on vaccinations, are ineffective. Therefore, new strategies need to be developed and implemented. This study aimed to evaluate whether supplementation with postbiotic products modulates the immune response in wild rabbits vaccinated against myxomatosis. For this purpose, two groups of rabbits were established: a control group fed with standard feed ad libitum from weaning (28 days) until two months of age, and a treated group, which was fed under the same conditions but supplemented with postbiotics (3 kg/Tm). All the studied rabbits were vaccinated against this disease during weaning. In addition, a blood samples were obtained from all animals immediately before vaccination and 30 days later, which allowed us to evaluate the level of antibodies against myxomatosis virus (ELISA detection) and the relative expression of gene encoding to cytokines related to the immune response (IL6, TNFα and IFNγ), at both times of the experience. Weight and length measurements were also taken at both times to calculate body index and mean daily gain (MDG). No statistically significant differences in growth parameters were observed. There were also no differences in the serological response among groups. However, a relative underexpression of gene codifying to TNFα (p-value = 0.03683) and a higher expression on IFNγ (p-value = 0.045) were observed in the treated group. This modulation in cytokines could lead to less severe lesions in wild rabbit naturally infected with myxomatosis virus.

Erysipelothrix rhusiopathiae is a relevant zoonotic infectious agent causing swine erysipelas (SE) in wild boar. In Portugal, there is no information on its occurrence. For this reason, this study aims to perform a first serosurvey of SE in hunted wild boars in Portugal. During the 2019/2020 hunting season, 111 sera from hunted wild boar were collected and analysed serologically in the laboratory with a commercial ELISA kit. No animals were eviscerated and examined after the hunt. The hunters took it all for private consumption. The results identified 18 animals that were exposed to SE, corresponding to a seroprevalence of 16.2% (95% CI: 19.9–24.4%). No statistical significance was observed on the effect of gender and age on seropositivity. However, wild boar hunted in Pinhel County, had five times more likely to be seropositivity (p-value < 0.05; OD = 5.4). Apart from its potential debilitating capacity and chronicity in the wild boar population, SE is also a very serious occupational zoonosis. Thus, the result of this first serosurvey in Portugal should raise awareness and alert competent national veterinary authorities and those involved in the hunting sector, especially hunters who directly handle these carcasses. Further studies should be conducted to better understand the role of wild boar as a reservoir and spillover of this disease to other animals and humans.

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.