Where's the Beef? Understanding Allergic Responses to Red Meat in Alpha-Gal Syndrome.

Alpha-gal syndrome (AGS) describes a collection of symptoms associated with IgE-mediated hypersensitivity responses to the glycan galactose-alpha-1,3-galactose (alpha-gal). Individuals with AGS develop delayed hypersensitivity reactions, with symptoms occurring >2 h after consuming mammalian ("red") meat and other mammal-derived food products. The mechanisms of pathogenesis driving this paradigm-breaking food allergy are not fully understood. We review the role of tick bites in the development of alpha-gal-specific IgE and highlight innate and adaptive immune cells possibly involved in alpha-gal sensitization. We discuss the impact of alpha-gal glycosylation on digestion and metabolism of alpha-gal glycolipids and glycoproteins, and the implications for basophil and mast cell activation and mediator release that generate allergic symptoms in AGS.

Bartonella spp. seroprevalence in tick-exposed Swedish patients with persistent symptoms.

BACKGROUND: Bartonella spp. are emerging pathogens transmitted by arthropod vectors, possibly including ticks. We have investigated signs of bartonellosis in Swedish patients with presumed tick-bite exposure and symptom duration of at least 6 months. METHODS: Serological testing for Bartonella henselae and Bartonella quintana was performed in 224 patients. Symptoms, tick exposure, evidence of co-infection and previous treatments were evaluated. Seropositive patients were compared to a matched group (twofold larger and negative serology) from the same study cohort. RESULTS: Seroprevalence was 7% for B. henselae and 1% for B. quintana, with one patient testing positive to both agents. Tick bites were reported by 63% of the patients in the seropositive group and 88% in the seronegative group and presumed tick exposure was more common in the seronegative group. Animal contact was equally common in both groups, along with reported symptoms. The most common symptoms were fatigue, muscular symptoms, arthralgia and cognitive symptoms. Exposure to co-infections was evenly distributed in the seropositive and seronegative groups. CONCLUSIONS: Antibodies to Bartonella were more common in this cohort of patients than in cohorts of healthy Swedish blood donors in previous studies but lower than those in blood donors from southern Europe. Positive Bartonella serology was not linked to any specific symptom, nor to (suspected) tick-bite exposure.

Analysis of selected serological parameters in patients with diagnosed Lyme borreliosis and in seropositive patients with no clinical symptoms.

OBJECTIVES: The aim of the study was to analyze some metalloproteinases, cytokines, and chemokines in LB patients and healthy seropositive subjects. The presence of IgM/IgG antibodies against specific Borreliella antigens was analyzed in the presence or absence of clinical manifestations of LB. MATERIAL AND METHODS: The study involved 38 patients diagnosed with LB and arthralgia and/or arthritis symptoms, and 57 foresters presenting no clinical symptoms of LB. The ELISA test was applied for general screening of anti-Borreliella IgM/IgG. Western blot was used for confirmatory diagnosis of LB for the positive and borderline results. Serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF, IL-8, CCL5, CXCL9/MIG, CCL2/MCP-1, CXCL10/IP-10 concentrations were measured with the use of the Human Cytometric CBA test. The concentration of MMP-2 and MMP-9 in the serum was determined with the use of ELISA tests. RESULTS: Analysis of the cytokines and chemokines revealed that only the concentration of IL-2 was significantly higher (2.4 pg/m; p=0.00641) in patients with LB symptoms than in the seropositive individuals (0.4 pg/ml). The MMP2 concentration was significantly higher (233.3 ng/ml; p=0.00294) in patients with clinical manifestations of LB than in those occupationally exposed to tick bites, but did not have anti-Borreliella antibodies (192.0 ng/ml). CONCLUSIONS: The presence of IgG antibodies against a number of Borreliella antigens and the differences in the IL-2 and MMP2 levels in seropositive or seronegative individuals and symptomatic LB patients, may indicate differences in the intensity of the immune response to the infection and, consequently, may induce development of clinical manifestations of the disease in seropositive and seronegative individuals.

The Infectivity Gene bbk13 Is Important for Multiple Phases of the Borrelia burgdorferi Enzootic Cycle.

Lyme disease is a multistage inflammatory disease caused by the spirochete Borrelia burgdorferi transmitted through the bite of an infected Ixodes scapularis tick. We previously discovered a B. burgdorferi infectivity gene, bbk13, that facilitates mammalian infection by promoting spirochete population expansion in the skin inoculation site. Initial characterization of bbk13 was carried out using an intradermal needle inoculation model of mouse infection, which does not capture the complex interplay of the pathogen-vector-host triad of natural transmission. Here, we aimed to understand the role of bbk13 in the enzootic cycle of B. burgdorferi. B. burgdorferi spirochetes lacking bbk13 were unable to be acquired by naive larvae fed on needle-inoculated mice. Using a capsule feeding approach to restrict tick feeding activity to a defined skin site, we determined that delivery by tick bite alleviated the population expansion defect in the skin observed after needle inoculation of Δbbk13 B. burgdorferi. Despite overcoming the early barrier in the skin, Δbbk13 B. burgdorferi remained attenuated for distal tissue colonization after tick transmission. Disseminated infection by Δbbk13 B. burgdorferi was improved in needle-inoculated immunocompromised mice. Together, we established that bbk13 is crucial to the maintenance of B. burgdorferi in the enzootic cycle and that bbk13 is necessary beyond early infection in the skin, likely contributing to host immune evasion. Moreover, our data highlight the critical interplay between the pathogen, vector, and host as well as the distinct molecular genetic requirements for B. burgdorferi to survive at the pathogen-vector-host interface and achieve productive disseminated infection.

The first human case of Rickettsia slovaca from Turkey.

A three year-old girl was admitted to our university hospital with fever, muscle and abdominal pain, and painful cervical lymph nodes after a tick bite on scalp. Rickettsia slovaca DNA was detected in eschar tissue taken from the bite site. This is the first clinical case of a R. slovaca infection reported from Turkey.

Ticking on Pandora's box: a prospective case-control study into 'other' tick-borne diseases.

BACKGROUND: Tick-borne pathogens other than Borrelia burgdorferi sensu lato - the causative agent of Lyme borreliosis - are common in Ixodes ricinus ticks. How often these pathogens cause human disease is unknown. In addition, diagnostic tools to identify such diseases are lacking or reserved to research laboratories. To elucidate their prevalence and disease burden, the study 'Ticking on Pandora's Box' has been initiated, a collaborative effort between Amsterdam University Medical Center and the National Institute for Public Health and the Environment. METHODS: The study investigates how often the tick-borne pathogens Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, Neoehrlichia mikurensis, spotted fever group Rickettsia species and/or tick-borne encephalitis virus cause an acute febrile illness after tick-bite. We aim to determine the impact and severity of these tick-borne diseases in the Netherlands by measuring their prevalence and describing their clinical picture and course of disease. The study is designed as a prospective case-control study. We aim to include 150 cases - individuals clinically suspected of a tick-borne disease - and 3 matched healthy control groups of 200 persons each. The controls consist respectively of a group of individuals with either a tick-bite without complaints, the general population and of healthy blood donors. During a one-year follow-up we will acquire blood, urine and skin biopsy samples and ticks at baseline, 4 and 12 weeks. Additionally, participants answer modified versions of validated questionnaires to assess self-reported symptoms, among which the SF-36, on a 3 monthly basis. DISCUSSION: This article describes the background and design of the study protocol of 'Ticking on Pandora's Box'. With our study we hope to provide insight into the prevalence, clinical presentation and disease burden of the tick-borne diseases anaplasmosis, babesiosis, B. miyamotoi disease, neoehrlichiosis, rickettsiosis and tick-borne encephalitis and to assist in test development as well as provide recommendations for national guidelines. TRIAL REGISTRATION: NL9258 (retrospectively registered at Netherlands Trial Register, trialregister.nl in in February 2021).

Coinfection of Ixodes scapularis (Acari: Ixodidae) Nymphs With Babesia spp. (Piroplasmida: Babesiidae) and Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) in Wisconsin.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is endemic and widespread in Wisconsin. Research in the northeastern United States has revealed a positive association between Babesia microti, the main pathogen that causes babesiosis in humans, and Bo. burgdorferi in humans and in ticks. This study was conducted to examine associations between the disease agents in the Upper midwestern United States. Ixodes scapularis Say nymphs (N = 2,858) collected between 2015 and 2017 from nine locations in Wisconsin were tested for Babesia spp. and Borrelia spp. using real-time PCR. Two species of Babesia were detected; Ba. microti and Babesia odocoilei (a parasite of members of the family Cervidae). Prevalence of infection at the nine locations ranged from 0 to 13% for Ba. microti, 11 to 31% for Bo. burgdorferi sensu stricto, and 5.7 to 26% for Ba. odocoilei. Coinfection of nymphs with Bo. burgdorferi and Ba. odocoilei was detected in eight of the nine locations and significant positive associations were observed in two of the eight locations. The prevalence of nymphal coinfection with both and Bo. burgdorferi and Ba. microti ranged from 0.81 to 6.5%. These two pathogens were significantly positively associated in one of the five locations where both pathogens were detected. In the other four locations, the observed prevalence of coinfection was higher than expected in all but one site-year. Clinics and healthcare providers should be aware of the association between Ba. microti and Bo. burgdorferi pathogens when treating patients who report tick bites.

Induced Transient Immune Tolerance in Ticks and Vertebrate Host: A Keystone of Tick-Borne Diseases?

Ticks and tick transmitted infectious agents are increasing global public health threats due to increasing abundance, expanding geographic ranges of vectors and pathogens, and emerging tick-borne infectious agents. Greater understanding of tick, host, and pathogen interactions will contribute to development of novel tick control and disease prevention strategies. Tick-borne pathogens adapt in multiple ways to very different tick and vertebrate host environments and defenses. Ticks effectively pharmacomodulate by its saliva host innate and adaptive immune defenses. In this review, we examine the idea that successful synergy between tick and tick-borne pathogen results in host immune tolerance that facilitates successful tick infection and feeding, creates a favorable site for pathogen introduction, modulates cutaneous and systemic immune defenses to establish infection, and contributes to successful long-term infection. Tick, host, and pathogen elements examined here include interaction of tick innate immunity and microbiome with tick-borne pathogens; tick modulation of host cutaneous defenses prior to pathogen transmission; how tick and pathogen target vertebrate host defenses that lead to different modes of interaction and host infection status (reservoir, incompetent, resistant, clinically ill); tick saliva bioactive molecules as important factors in determining those pathogens for which the tick is a competent vector; and, the need for translational studies to advance this field of study. Gaps in our understanding of these relationships are identified, that if successfully addressed, can advance the development of strategies to successfully disrupt both tick feeding and pathogen transmission.

Scalp eschar and neck lymphadenopathy by Rickettsia slovaca after Dermacentor marginatus tick bite case report: multidisciplinary approach to a tick-borne disease.

BACKGROUND: Scalp Eschar and Neck LymphAdenopathy after Tick bite is a zoonotic non-pathogen-specific disease most commonly due to Rickettsia slovaca and Rickettsia raoultii. Diagnosis is mostly based only on epidemiological and clinical findings, without serological or molecular corroboration. We presented a clinical case in which diagnosis was supported by entomological identification and by R. slovaca DNA amplifications from the tick vector. CASE PRESENTATION: A 6-year-old child presented with asthenia, scalp eschar and supraclavicular and lateral-cervical lymphadenopathy. Scalp Eschar and Neck LymphAdenopathy After Tick bite syndrome following a Dermacentor marginatus bite was diagnosed. Serological test on serum revealed an IgG titer of 1:1024 against spotted fever group rickettsiae, polymerase chain reaction assays on tick identified Rickettsia slovaca. Patient was successfully treated with doxycycline for 10 days. CONCLUSIONS: A multidisciplinary approach including epidemiological information, clinical evaluations, entomological identification and molecular investigations on tick, enabled proper diagnosis and therapy.

First identification of Anaplasma phagocytophilum in both a biting tick Ixodes nipponensis and a patient in Korea: a case report.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum. To date, there have been no reported cases of A. phagocytophilum infection found in both the biting tick and the patient following a tick bite. CASE PRESENTATION: An 81-year-old woman presented with fever following a tick bite, with the tick still intact on her body. The patient was diagnosed with HGA. The tick was identified as Ixodes nipponensis by morphological and molecular biological detection methods targeting the 16S rRNA gene. The patient's blood was cultured after inoculation into the human promyelocytic leukemia cell line HL-60. A. phagocytophilum growth was confirmed via culture and isolation. A. phagocytophilum was identified in both the tick and the patient's blood by Anaplasma-specific groEL- and ankA-based nested polymerase chain reaction followed by sequencing. Moreover, a four-fold elevation in antibodies was observed in the patient's blood. CONCLUSION: We report a case of a patient diagnosed with HGA following admission for fever due to a tick bite. A. phagocytophilum was identified in both the tick and the patient, and A. phagocytophilum was successfully cultured. The present study suggests the need to investigate the possible incrimination of I. nipponensis as a vector for HGA in Korea.

Asymptomatic-anaplasmosis confirmation using genetic and serological tests and possible coinfection with spotted fever group Rickettsia: a case report.

BACKGROUND: Anaplasmosis is an emerging acute febrile disease that is caused by a bite of an Anaplasma phagocytophilum-infected hard tick. As for healthy patients, reports on asymptomatic anaplasmosis resulting from such tick bites are rare. CASE PRESENTATION: A 55-year-old female patient visited the hospital with a tick bite in the right infraclavicular region. The tick was suspected to have been on the patient for more than 10 days. PCR and an indirect immunofluorescence assay (IFA) were performed to identify tick-borne infectious diseases. The blood sample collected at admission yielded a positive result in nested PCR targeting Ehrlichia- or Anaplasma-specific genes groEL and ankA. Subsequent sequencing confirmed the presence of A. phagocytophilum, and seroconversion was confirmed by the IFA involving an A. phagocytophilum antigen slide. PCR detected no Rickettsia-specific genes [outer membrane protein A (ompA) or surface cell antigen 1 (sca1)], but seroconversion of spotted fever group (SFG) rickettsiosis was confirmed by an IFA. CONCLUSIONS: This study genetically and serologically confirmed an asymptomatic A. phagocytophilum infection. Although SFG rickettsiosis was not detected genetically, it was detected serologically. These findings indicate the possibility of an asymptomatic coinfection: anaplasmosis plus SFG rickettsiosis. It is, therefore, crucial for clinicians to be aware of potential asymptomatic anaplasmosis following a tick bite.

Assessment of Borrelia miyamotoi in febrile patients and ticks in Alsace, an endemic area for Lyme borreliosis in France.

BACKGROUND: Borrelia miyamotoi is a relapsing fever Borrelia species transmitted by ticks of the Ixodes ricinus complex. Human disease caused by B. miyamotoi was first described in Russia and later in the USA and Japan. Additionally, five cases of meningoencephalitis in immunocompromised patients and one case in an apparently immunocompetent patient were described. METHODS: We investigated the presence of B. miyamotoi in I. ricinus nymphs and in patients suspected of human granulocytic anaplasmosis, in Alsace (France), an endemic area for I. ricinus ticks and Lyme borreliosis, using direct (PCR) and indirect diagnosis (glycerophosphoryldiester-phosphodiesterase (GlpQ) serology). RESULTS: Borrelia miyamotoi was found in 2.2% of 4354 ticks collected between 2013 and 2016. None of the 575 blood samples, collected from the patients suspected of HGA, was found positive for B. miyamotoi by PCR. Acute and late sera from 138 of these 575 patients were available. These paired sera were tested for IgM and IgG antibodies against the B. miyamotoi GlpQ antigen. A total of 14 out of 138 patients had at least one positive parameter (i.e. anti-GlpQ IgG and/or IgM). One patient seroconverted for IgG, and three had isolated IgM in the acute serum. These three patients were treated with doxycycline which could have prevented seroconversion. After reviewing clinical data and other biological tests performed, co-exposure among different microorganisms vectored by ticks or serological cross-reactivity could not be ruled out in these different cases. One patient had persistent IgG, which strongly suggests previous exposure to B. miyamotoi. CONCLUSIONS: Humans can be exposed to B. miyamotoi through tick bites in Alsace. We present serological data for possible B. miyamotoi exposure or infection of patients with fever after tick bite. Future studies should determine the incidence, clinical course and burden of this emerging tick-borne disease in other parts of Western Europe.

A rickettsia infection from Saudi Arabia.

We here present a young patient who developed fever, headache, maculopapular skin rash and an eschar 3 days following a tick bite. Serology for Rickettsia was consistent with acute spotted fever group (SFG) infection. He had a dramatic response to doxycycline. Vectors for rickettsia transmission are shown in a number of studies from Saudi Arabia while human serological studies from Oman revealed that these infections are common among the rural population. Surprisingly, despite the existence of potential vectors, this disease is rarely reported from the Arab Gulf countries. To the best of our knowledge this is the first case report of SFG acquired within Saudi Arabia. SFG should be considered in the differential diagnosis of febrile illness and exposure history.

Serologic evidence of rickettsial diseases associated with tick bites in workers of urban veterinary clinics.

BACKGROUND & OBJECTIVES: Rickettsial and other zoonotic diseases are a latent risk for workers of veterinary clinics. The objective of this study was to evaluate the prevalence and the associated risk factors of parasitosis caused by Rhipicephalus sanguineus, and to estimate the seroprevalence of rickettsial diseases in workers of urban veterinary clinics of Juárez city, México. METHODS: The participants of the study were recruited from 63 private veterinary clinics and hospitals. The serological analysis of the blood samples collected was carried out using immunofluorescence assay (IFA). The statistical analysis for prevalences, risk factors, and correlation was performed with the SAS program. RESULTS: In total, 167 veterinary workers were included in the study. The prevalence of tick bites was 40% (67/167), and the risk factors associated with the occurrence of bites included the activities performed in the clinic and the number of labour hours spent per week. About 21% (35/167) of participants were seropositive to R. rickettsii, 28% (47/167) to Ehrlichia chaffeensis, and 24% (40/167) to Anaplasma phagocytophilum. A correlation was observed between: the number of workers in the clinics and the proportion of tick bites (r2 = 0.865); the prevalence of bites and the seropositivity of the participants to at least one pathogen (r2 = 0.924); and the number of bites per individual and infection to pathogens (r2 = 0.838). INTERPRETATION & CONCLUSION: Workers in urban veterinary clinics are highly exposed to tick bites and, therefore, to the diseases they transmit. Hence, it is important to implement prevention measures and perform constant monitoring of these diseases.

Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses.

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

Cellulitis of the face associated with SENLAT caused by Rickettsia slovaca detected by qPCR on scalp eschar swab sample: An unusual case report and review of literature.

BACKGROUND: Tick-borne rickettsioses are infectious diseases caused by obligate intracellular Gram-negative bacteria belonging to the spotted fever groupof Rickettsia. METHODS: We describe an unusual case of SENLAT (Scalp eschar and neck lymphadenopathy after tick bite), caused byRickettsia slovaca, associated with a cellulitis of the face in a 70-year-old woman, and diagnosed using qPCR on a scalp eschar swab. We review the literature regarding cases of SENLAT-associated-cellulitis and case of SENLAT diagnosed by qPCR on scalp eschar swabs. RESULTS: We found only one previous report of SENLAT associated with a cellulitis of the face. It was a nine-year-old French girl diagnosed by seroconversion for Rickettsia sp. Our review of the literature showed that qPCR on eschar swab samples is a less invasive method than performing cutaneous biopsy of the eschar and has good sensitivity and specificity (90% and 100%, respectively). CONCLUSIONS: We report the second case of cellulitis of the face associated with the SENLAT syndrome. Detection of Rickettsia by qPCR on swab sample of the scalp eschar is a simple, noninvasive technique allowing rapid diagnosis and treatment when SENLAT is suspected.

Azithromycin nanocrystals for dermal prevention of tick bite infections.

Azithromycin was optimized as nanocrystals with a drug content of 10.0 % (w/w) and a surfactant D-α -tocopheryl polyethylenglycol 1000 succinate (TPGS) content of 1.0 % (w/w) using bead milling for 10 min. The photon correlation spectroscopy (PCS) diameter of the bulk population was 189 nm, laser diffraction (LD) diameter 90 % was 370 nm. Spherical morphology of the optimal nanocrystals was observed by transmission electron microscope (TEM). They were stable over 1 year of storage at 4 °C with the particle size within the nanometer range which was confirmed by PCS, LD and light microscope. An acceptable physical stability of 2 years was also obtained when stored at 4 °C. No microbial attack to the nanocrystals was observed before 3 years storage at 4 °C. The saturation solubility of the nanocrystals was up to triple compared to the raw drug powder (RDP) in water. When incorporated into the gel base, highest penetration efficacy was achieved by the optimal nanocrystals compared to 1) the clinically effective ethanol-solution-gel, 2) the gel with propylene glycol and 3) the gel with RDP in the ex vivo porcine ear penetration study. Even though propylene glycol improved saturation solubility of nanocrystals, it could not bring benefit to nanocrystals in the penetration study. Based on these optimized azithromycin nanocrystals, topical administration for enhanced dermal bioavailability of azithromycin seems to be feasible.

Seroprevalence of Borrelia burgdorferi, B. miyamotoi, and Powassan Virus in Residents Bitten by Ixodes Ticks, Maine, USA.

We conducted a serosurvey of 230 persons in Maine, USA, who had been bitten by Ixodes scapularis or I. cookei ticks. We documented seropositivity for Borrelia burgdorferi (13.9%) and B. miyamotoi (2.6%), as well as a single equivocal result (0.4%) for Powassan encephalitis virus.

Exploring the bacteriome in anthropophilic ticks: To investigate the vectors for diagnosis.

OBJECTIVE: The aim of this study was to characterize the bacterial microbiome of hard ticks with affinity to bite humans in La Rioja (North of Spain). METHODS: A total of 88 adult ticks (22 Rhipicephalus sanguineus sensu lato, 27 Haemaphysalis punctata, 30 Dermacentor marginatus and 9 Ixodes ricinus) and 120 I. ricinus nymphs (CRETAV collection, La Rioja, Spain), representing the main anthropophilic species in our environment, were subjected to a metagenomic analysis of the V3-V4 region of the 16S rRNA gene using an Illumina MiSeq platform. Data obtained with Greengenes database were refined with BLAST. Four groups of samples were defined, according to the four tick species. RESULTS: Proteobacteria was the predominant phylum observed in all groups. Gammaproteobacteria was the most abundant class, followed by Alphaproteobacteria for R. sanguineus, H. punctata and D. marginatus but the relative abundance of reads for these classes was reversed for I. ricinus. This tick species showed more than 46% reads corresponding to 'not assigned' OTUs (Greengenes), and >97% of them corresponded to 'Candidatus Midichloriaceae' using BLAST. Within Rickettsiales, 'Candidatus Midichloria', Rickettsia, Ehrlichia, 'Candidatus Neoehrlichia' and Wolbachia were detected. I. ricinus was the most alpha-diverse species. Regarding beta-diversity, I. ricinus and H. punctata samples grouped according to their tick species but microbial communities of some R. sanguineus and D. marginatus specimens clustered together. CONCLUSIONS: The metagenomics approach seems useful to discover the spectrum of tick-related bacteria. More studies are needed to identify and differentiate bacterial species, and to improve the knowledge of tick-borne diseases in Spain.