The development of parameters to estimate the risk of transmission of infections is one of the objectives of entomological surveillance, included in the National Plan for the Prevention, Surveillance and Control of Vector-Borne Diseases. This study aims to assess the utility of the infection prevalence (IP) of Rickettsia spp., Borrelia burgdorferi, and Crimean-Congo haemorrhagic fever virus (CCHFV) in ticks in Spain, as an indicator of the risk of infection from a tick bite and for temporal and geographical comparisons.
MethodsThe IP and its 95% confidence interval (95% CI) were calculated for the different microorganism-tick pairs, using data published until April 2024, obtained through a systematic literature review. Studies that included pathogen detection using molecular methods in ticks collected from vegetation (questing ticks) were selected.
Results581 articles were reviewed, and 20 were selected. The IP of Rickettsia spp. in Dermacentor marginatus was 84.4%, in D. reticulatus 45.9%, and in Rhipicephalus sanguineus 21.2%, with some Rickettsia species causing DEBONEL (R. raoultii and R. slovaca) and MSF-like illness (R. massiliae) being the most frequent. The IP of Borrelia burgdorferi sensu lato in Ixodes ricinus was 5.9%. The IP of CCHFV in Hyalomma spp was 0.3%.
ConclusionsThe IP varies for each pathogen-tick pair, from very high for Rickettsia in Dermacentor, to relatively low for Borrelia in Ixodes and CCHFV in Hyalomma. The infection prevalence in ticks can be a useful indicator, along with other factors, for estimating the risk of infection from a tick bite and conducting population risk assessments in Spain.
El desarrollo de parámetros para estimar el riesgo de transmisión de infecciones es uno de los objetivos de la vigilancia entomológica, contemplado en el Plan Nacional de Prevención, Vigilancia y Control de las enfermedades transmitidas por vectores. El objetivo de este estudio es valorar la utilidad de la prevalencia de infección (PI) de Rickettsia spp., Borrelia burgdorferi y virus de la fiebre hemorrágica Crimea-Congo (VFHCC) en garrapatas de España, como indicador de riesgo de infección ante picadura y para comparaciones temporales y geográficas.
MétodosSe calculó la PI y su intervalo de confianza al 95% (IC95%) para las distintas parejas microorganismo-garrapata, con datos publicados hasta abril de 2024, obtenidos mediante revisión bibliográfica sistemática. Se seleccionaron estudios con detección de patógenos por métodos moleculares en garrapatas recogidas en vegetación.
ResultadosSe revisaron 581 artículos y se seleccionaron 20. La PI de Rickettsia spp. en Dermacentor marginatus fue 84,4%, en D. reticulatus 45,9%, y en Rhipicephalus sanguineus 21,2%, siendo algunas especies de rickettsia causales de DEBONEL (R. raoultii y R. slovaca) y de rickettsiosis FEM-like (R. massiliae) las más frecuentes. La PI de Borrelia burgdorferi s.l. en Ixodes ricinus fue 5,9%. La PI de VFHCC en Hyalomma spp fue 0,3%.
ConclusionesLa PI es variable para cada pareja patógeno-garrapata, desde muy elevada para Rickettsia en Dermacentor, a relativamente baja para Borrelia en Ixodes y VFHCC en Hyalomma. La PI puede ser un indicador útil, junto a otros factores, para estimar el riesgo de infección ante una picadura y realizar evaluaciones de riesgo poblacional en España.
Ticks are a superfamily of haematophagous mites (arthropods of the class Arachnida, subclass Acari) important in terms of public health because they act as vectors of pathogens which can be transmitted to humans if they are bitten. The risk of transmission depends on various factors, including the biology of the vector, its habitat and the potential hosts.
Ticks of public health concern belong to two families: Ixodidae (hard ticks) and Argasidae (soft ticks). Argasidae usually live inside nests or burrows, and have little contact with humans. Species of the family Ixodidae tend to live in vegetation, a factor that contributes to the fact that this group includes most of the ticks that can transmit pathogens to humans.1
These arthropods have four stages: egg, larva, nymph and adult. After hatching, the tick needs to feed on the blood of a vertebrate before moving on to the next stage, or to lay eggs in the case of an adult female. When they ingest blood from an animal infected with a given pathogen, they can acquire the pathogen (vector competence) and transmit it to new hosts (vector capacity) during subsequent ingestion of blood.1 Immature stages normally feed on small hosts such as rodents, lagomorphs, birds or even reptiles. Adults, however, tend to feed on larger mammals (carnivores or ungulates). At the same time, certain genera and species are generalists, while others are specialists, meaning they have an affinity for certain hosts and rarely bite other animal families. This means that not all ticks have the same degree of anthropophilia; in other words, they do not have the same affinity for biting humans for food.2,3 Hosts can also play an important role in the movement and distribution of ticks and the diseases they may transmit. Other vector-dependent factors involved in pathogen transmission include the duration of the ingestion of blood in each species,4 the trans-stage and transovarial transmission capacity of the pathogens, and, particularly, the percentage of infected ticks in a given area and the vectorial capacity of the different species.1 Factors affecting biting risk are the distribution, life cycle length and habitats of each tick species, the likelihood of exposure of the population to these habitats and the periods of the year of highest activity for the different evolutionary stages.
Climate change and some human actions, such as animal transport and changes in land use which favour the proliferation of some wild animals, are causing changes in the distribution and population of ticks worldwide, such as the increase of Hyalomma lusitanicum populations in areas where rabbits and some ungulates such as wild boar are becoming more abundant.5 These phenomena, combined with other factors, are creating new scenarios in which an emergence or re-emergence of tick-borne diseases can be expected.6
In Spain there is a great diversity of tick species, distributed heterogeneously depending on environmental factors such as climate and the presence of hosts necessary to complete their life cycle, with a much smaller number of species of public health concern, as only some may be infected and capable of transmitting diseases through their bite.
In April 2023, the Spanish Ministry of Health published the National Plan for the Prevention, Surveillance and Control of Vector-Borne Diseases (hereafter referred to as the Plan),7 to which in July 2024 a third part was added focusing on tick-borne diseases.8 Some of these diseases are considered emerging, such as Crimean-Congo haemorrhagic fever (CCHF); others, such as Borrelia miyamotoi relapsing fever and tick-borne encephalitis have the potential for emergence; and lastly, some are endemic, such as Lyme borreliosis and rickettsial diseases (Mediterranean exanthematic fever [MEF], MEF-type rickettsial disease, Dermacentor-borne-necrosis-erythema-lymphadenopathy [DEBONEL]/tick-borne lymphadenopathy [TIBOLA]). The objectives of the Plan7,8 included "increasing entomological surveillance of ticks to detect their presence and developing other entomological parameters to estimate the risk of disease transmission". We designed this study with these objectives in mind and taking into account the diseases of greatest interest in our setting and included in the Plan.
The aim was to assess the utility of estimating at a national level the prevalence of infection (PI) in ticks by pathogens of public health concern, as an indicator of the risk, combined with other factors, of rickettsiosis, CCHF or Lyme disease if bitten, and to be able to make temporal and geographical comparisons enabling population risk assessments in Spain.
Material and methodsFirst, we carried out a systematic review of the literature published up to 18 April 2024 in English or Spanish in the PubMed/MEDLINE database using a Boolean combination, including terms related to the pathogen or disease being studied, the vector and the country. The strategies were: ricket* AND tick* AND (Spain OR Spanish OR Iberi*) for the study of rickettsiosis (crimea* OR cchf) AND tick* AND (Spain OR Spanish OR Iberi*) for CCHF, and (borrel* OR Lyme) AND tick* AND (Spain OR Spanish OR Iberi*) for Lyme disease. Using the results of the searches, an initial screening was done by title and abstract, and a second screening was done with a full review of some articles (Fig. 1). Studies had to meet the following inclusion criteria:
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Original articles from studies carried out in mainland Spain and the Balearic Islands.
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Sampling of ticks, nymphs and/or adults, on vegetation.
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Determination of pathogens by molecular methods: Rickettsia spp. in Ixodidae, CCHF virus in Hyalomma spp. and Borrelia spp. in Ixodes ricinus.
The first review was carried out simultaneously by two different people. A third researcher reviewed the articles where there were doubts. From the articles that met the inclusion criteria, the following information was extracted for each pathogen studied: Autonomous Region(s) of sampling (in Spain); year of start and year of completion; method of collection; genus, species and stages of ticks collected in vegetation; number of ticks, by genus and species, analysed by molecular methods for the pathogens of interest and number of positive specimens; in batch analysis, number of specimens per batch, total number of ticks analysed per batch and number of positive batches. For Rickettsia spp. and Borrelia spp., if identification was performed, species and/or subspecies of the pathogen detected by sequencing or other molecular techniques, and total number per tick species.
We calculated prevalence of infection (PI) for the pathogens in vector ticks for each pathogen-vector pair, as the number of positive ticks over the number of ticks tested (positive ticks/tested ticks). In batch studies, a positive batch was interpreted as one positive tick out of the total number of ticks in the batch (minimum prevalence of infection). Results are expressed as a percentage with a 95% confidence interval (95% CI) for a proportion, which was calculated using the Wilson scoring method without continuity correction, using a confidence interval calculator Excel spreadsheet.9
ResultsA total of 581 articles were detected after applying the literature search strategies. After evaluation of those that met the inclusion criteria, screening by title and abstract and full reading of the pre-selected articles, nine were finally included for Rickettsia spp. in hard ticks, three for CCHF virus in ticks of the genus Hyalomma, and eight for Borrelia spp. in I. ricinus. The algorithm is shown in Fig. 1.
Prevalence of Rickettsia spp. infection in hard ticksFrom the nine publications that met the predefined inclusion criteria,10–18 information was extracted for the analysis of a total of 4340 ticks of various anthropophilic species of the genera Dermacentor, Ixodes, Rhipicephalus, Haemaphysalis and Hyalomma, collected from vegetation from 2000 to 2021 in seven autonomous regions (Andalusia, Asturias, Castile-La Mancha, Galicia, La Rioja, Madrid Region and the Basque Country).
All studies determined positivity by individual analysis of nymphs and/or adults of the species included, but one study included batches (370 nymphs, with five per batch). With this, the estimated PI over the period 2000–2021 was 16.8% (95% CI: 15.7–17.9). The Ixodid tick species with the highest prevalence found were: Dermacentor marginatus 84.4%, Dermacentor reticulatus 45.9%, Rhipicephalus sanguineus group 21.2% and I. ricinus 14.6%. The PI for Rickettsia spp. for each species and the corresponding 95% CI are shown in Fig. 2.
Prevalence of Rickettsia spp. infection in Ixodidae tick species collected from vegetation. Spain, 2000–2021.
D.: Dermacentor; 95% CI: 95% confidence interval; PI: prevalence of infection; Rh.: Rhipicephalus.
* Rh. sanguineus group: includes 95 Rh. sanguineus sensu lato, 8 Rh. pusillus and 15 Rh. turanicus.
^ Includes nymphs analysed in batches of five specimens per batch (300 I. ricinus, 20 Hae. punctata and 50 Hae. cocinna).
The most commonly found Rickettsia species out of the total number of Ixodidae analysed (4340) and with species identification (616) were: Rickettsia raoultii (6.1%), Rickettsia slovaca (3.2%) and Candidatus Rickettsia rioja (3.2%), mainly found in D. marginatus, D. reticulatus and I. ricinus; followed by Rickettsia monacensis (1.1%) and Rickettsia massiliae (0.6%), detected in Rh. sanguineus and I. ricinus. Co-infection was detected in 46 specimens, the most common being R. raoultii/Ca. R. rioja, in 40 I. ricinus. The PI by Rickettsia species and tick species in which detection was performed are shown in Fig. 3.
Prevalence of Rickettsia species infection detected in Ixodidae ticks collected from vegetation. Spain, 2000–2021.
Ca: Candidatus; D: Dermacentor; H: Hyalomma; Hae: Haemaphysalis; PI: prevalence of infection; Rh: Rhipicephalus.
¥ In D. reticulatus, species identification was performed on 30 of the 56 ticks with detection of Rickettsia spp.
* Rh. sanguineus group: includes 95 Rh. sanguineus sensu lato, 8 Rh. pusillus and 15 Rh. turanicus.
^ Includes 300 nymphs of I. ricinus and 20 of Hae. punctata in batches of 5.
˭ Includes co-infection of R. massiliae with R. monacensis, R. aeschlimanii and Rickettsia sp.
'' Includes 15 Hae. sulcata and 3 Hae. punctata with detection of R. hoogstraalii (endosymbiont), one I. ricinus with R. felis, and 20 without species identification (R. sp.).
Three articles19–21 were included in the CCHF virus positivity study, reviewing data from 2,602 ticks (91.6% of them H. lusitanicum) collected on vegetation from 2011 to 2018 (68.8% in the period 2016–2018) in nine of Spain's autonomous regions. The largest number of ticks tested came from Andalusia (1335), Castile-La Mancha (547) and Madrid (438). The rest came from Aragón (128), Murcia (99), Extremadura (41), La Rioja (10), Castile and León (3) and Navarre (1). Of the 219 Hyalomma marginatum analysed, 58.4% and 35.6% were collected in 2011 in Aragón and Castile-La Mancha respectively.
Batch testing was carried out in one of the three studies (1791 H. lusitanicum, in batches of three), with positivity in seven of the batches. The estimated PI for ticks of the genus Hyalomma was 0.3% (95% CI 0.1−0.6); H. marginatum 0% (95% CI 0–1.7) and H. lusitanicum 0.3% (95% CI 0.1−0.6).
Prevalence of Borrelia spp. infection in Ixodes ricinusWe analysed data from a total of 6222 ticks of the species I. ricinus from the eight selected studies.12,16,18,22–26 These ticks were collected on vegetation in the period from 1995 to 2021 in six autonomous regions (Asturias, Cantabria, Galicia, La Rioja, Navarre and the Basque Country).
Half of the studies conducted batch analyses, with a total of 3728 I. ricinus, 382 adults and 3346 nymphs, in batches of three to 30 specimens. The PI for Borrelia spp. was estimated at 6.2% (95% CI 5.6–6.8), and for Borrelia sensu lato at 5.9% (95% CI 5.3–6.5).
The most common genospecies were Borrelia garinii and Borrelia afzelii, both species considered to be pathogenic. B. miyamotoi was detected in 0.3% of the specimens tested. The PI of the detected Borrelia species and subspecies, and their percentage distribution, are shown in Fig. 4.
Prevalence of infection (columns) and distribution (sectors) of Borrelia species detected in Ixodes ricinus collected from vegetation. Spain, 1995–2021.
B.: Borrelia; PI: prevalence of infection.
In B. burgdorferi sensu lato, species are represented which are considered to be: * pathogenic; ** of low pathogenicity; *** unproven pathogenicity.
In this study, we estimated the prevalence of infection by certain pathogens in different species of ticks of the family Ixodidae which may act as vectors of diseases of importance to human health in Spain, in order to provide a useful parameter for the estimation of individual risk, in case of being bitten, and of population risk. We reviewed data from studies of pathogens in hard ticks with distribution all over Spain.27–29 The exclusive selection of ticks collected from vegetation was made for two main reasons. The first was that most of the species of concern to health are either in the soil, waiting for a host, or actively searching for a host, and are therefore more likely to come into contact with people. The second was to avoid testing for fed ticks, as this could overestimate the PI, because the microorganism has been acquired from the animal after feeding. Two factors were taken into account for the inclusion of pathogen-vector pairs in this review. Firstly, that they are tick species which bite humans to a greater or lesser degree, ranging from those known to be more anthropophilic, such as I. ricinus, to others with a low rate of documented bites, such as Haemaphysalis spp.30 Secondly, we included tick species with vectorial capacity, i.e. those with evidence of transmission of the pathogen to humans, regardless of whether there were previously described cases here in Spain.
Individual and batch studies were used to calculate the PI. In batch studies, the minimum prevalence of infection was used, where it is assumed that one positive batch equals one positive sample or one positive specimen out of the total number of samples included in the batch.31 This method is very suitable for low prevalence situations (less than 2%),32 such as CCHF virus infection in Hyalomma spp., but may underestimate prevalence if prevalence is higher, as in the case of Rickettsia spp. In any case, this pathogen was detected individually in 91.5% of the ticks in the studies analysed.
The results of this study may be useful in helping to estimate the risk of infection from a tick bite. Identification of the genus and, if possible, the species of the captured tick is also of great importance, as the presence or prevalence of pathogens varies from species to species. For example, for a bite where no further information is available, the PI of 16.8% for Rickettsia spp. may indicate the risk of infection in the person bitten, taking into account other factors, such as how long the tick has remained attached and whether it has been handled by the person in an attempt to remove it, and factors dependent on the tick species (vectorial capacity) and the person (individual susceptibility). If the genus involved is known to be Dermacentor spp. and, better still, the bite is known to be due to the species D. marginatus or D. reticulatus, the risk estimate will be higher, and mainly due to infection by R. raoultii and R. slovaca, pathogens related to diseases such as DEBONEL/TIBOLA.33 Similarly, the PI of 21.2% for Rickettsia spp. in the R. sanguineus group may indicate the risk of infection in the event of being bitten by the species included, and that within these, R. massiliae, the causative agent of MEF-type rickettsiosis, is the most common Rickettsia.33 In H. lusitanicum, a species considered to be not very anthropophilic,3 the estimated PI for CCHF virus was 0.3% (ranging from 0.1 to 0.6%). This prevalence is much lower than that found in other studies, which included ticks obtained from animals and in areas of known higher virus circulation.21 Very few specimens of H. marginatum, the main known vector of CCHF, were included in this study, and this may be because it is a species that quickly attaches onto hosts, including humans, making it difficult to find in soil. In the case of I. ricinus, a species considered more anthropophilic,34 especially the nymphs, the PI of Borrelia burgdorferi sensu lato was 5.9%, with the most common subspecies being B. garinii, followed by B. afzelii, both of which cause Lyme disease,35 and Borrelia valaisiana, which is less pathogenic.
This study has a series of limitations. On the one hand, the variability in the methodology used to collect specimens in the different studies analysed may have produced variations in the results from one study to another. The uneven distribution of the samples, as well as the total sample size, did not allow estimates to be made for different geographical locations. In Hyalomma, the estimated PI for CCHF virus may be underestimating the risk of infection, given the low proportion of H. marginatum relative to H. lusitanicum included, and the fact that a large number of samples came from areas considered low risk. Another consideration is that the data in this study do not allow for time series analysis. To avoid these limitations, more systematic studies would be necessary, such as those proposed at a national level in entomological surveillance, in addition to the use of standardised protocols for sampling and for the study of pathogens in ticks. Other requirements are standardisation of the variables used in entomological surveillance and the establishing of indicators which enable spatial and temporal comparisons.
In conclusion, taken with other factors, the prevalence of infection in different pathogen-tick pairs may be a useful indicator for estimating the risk of infection in the event of being bitten. The results of this study can also provide us with a basis for establishing temporal and geographical comparisons with data derived from entomological surveillance, enabling population risk assessments in Spain which can serve as a guide for implementing appropriate prevention and control.
FundingThe authors declare that they received no funding to conduct this study.
The authors declare that there are no conflicts of interest.
We would like to thank Agustín Estrada Peña (SPVECTORSURV [Scaling up One Health vector-borne zoonotic diseases monitoring and surveillance in Spain] Project, Spanish Ministry of Health), Sonia Olmeda (Universidad Complutense de Madrid), Félix Valcárcel (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria [Spanish Institute of Agricultural and Food Research and Technology]), María Paz Sánchez-Seco, Ana Vázquez and Anabel Negredo (Centro Nacional de Microbiología, [Spanish Microbiology Centre] Instituto de Salud Carlos III [Carlos III Health Institute]) for their scientific advice.
