Alpha-Gal, epitope responsible for allergy to red meat, in the Mediterranean tick Hyalomma lusitanicum.

Alpha-Gal/α-Gal is an oligosaccharide produced by non-primate mammals. Humans have developed an immune response mediated by anti-α-Gal antibodies that can trigger an allergic reaction and cause anaphylaxis. In recent years, cases of patients with delayed allergic reaction to mammalian meat have been reported worldwide. In Spain, these cases have been related to the species Ixodes ricinus L. (Ixodida: Ixodidae), whose distribution is located in the north of the country. In this work, the presence of α-Gal in water-soluble extracts from samples of salivary glands and digestive tracts of Hyalomma lusitanicum Koch (Ixodida: Ixodidae) both engorged and collected from vegetation were studied. The presence of that epitope was confirmed by the presence of reactive proteins of >250 kDa in both samples. The highest concentrations of α-Gal were detected in salivary glands. Neither sex nor diet influenced the concentration of α-Gal, which seems to indicate its endogenous production and its possible inoculation to the host during tick feeding.

Emerging Hyalomma lusitanicum: From identification to vectorial role and integrated control.

In the Mediterranean basin, the tick species Hyalomma lusitanicum Koch stands out among other species of the Hyalomma genus due to its wide distribution, and there is great concern about its potential role as a vector and/or reservoir and its continuous expansion to new areas because of climate warming and human and other animal movements. This review aims to consolidate all the information on H. lusitanicum, including taxonomy and evolution, morphological and molecular identification, life cycle, sampling methods, rearing under laboratory conditions, ecology, hosts, geographical distribution, seasonality, vector role and control methods. The availability of adequate data is extremely relevant to the development of appropriate control strategies in areas where this tick is currently distributed as well as in new areas where it could become established in the near future.

Acaricidal and Repellent Effects of Essential Oils against Ticks: A Review.

Tick control is a priority in order to prevent the transmission of vector-borne diseases. Industrial chemical acaricides and repellents have been the most efficient tools against hard ticks for a long time. However, the appearance of resistances has meant the declining effectiveness of the chemicals available on the market. The trend today is to develop alternative control methods using natural products to replace nonefficient pesticides and to preserve the efficient ones, hoping to delay resistance development. Traditional in vitro evaluation of acaricidal activity or resistance to synthetic pesticides have been reviewed and they mainly focus on just one species, the one host tick (Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)). Recent reports have called for the standardization of natural product components, extraction techniques, and experimental design to fully discover their acaricidal potential. This study reviews the main variables used in the bibliography about the efficiency of natural products against ticks, and it proposes a unification of variables relating to ticks, practical development of bioassays, and estimation of ixodicidal activity.

Seasonal Dynamics of Fleas (Siphonaptera: Pulicidae, Ceratophyllidae and Leptopsyllidae) on Oryctolagus cuniculus in a Meso-Mediterranean Area of Central Spain.

Flea infestations of wild rabbits were examined monthly in central Spain in a meso-Mediterranean area for 5 yr. A total of 1,180 wild rabbits were trapped and 7,022 fleas were collected from them. Overall, the prevalence was 74.1% with a mean flea index of 5.95 fleas per rabbit. Four flea species were identified: Spilopsyllus cuniculi (Dale, 1878) was the most abundant species (accounting for 74.3% of fleas collected) followed by Xenopsylla cunicularis (Smit, 1957), Odontopsyllus quirosi (Gil Collado, 1934), and Nosopsyllus fasciatus (Bosc, 1800) (18.9, 6.7, and 0.1%, respectively). The highest prevalence was observed in S. cuniculi (48.6%) followed by X. cunicularis, O. quirosi, and N. fasciatus (34.3, 20.0, and 0.6%, respectively). Odontopsyllus quirosi and S. cuniculi were mainly collected from autumn to spring with the peak of infestation in winter, while X. cunicularis was mainly found from spring to autumn with maximum levels of infestation during the summer months. The relevance of these findings is discussed.

Seasonality of Coxiella burnetii among Wild Rabbits (Oryctolagus cuniculus) and the Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem.

(1) Background: Q fever is a worldwide zoonosis caused by Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs. Presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.

Transstadial Transmission from Nymph to Adult of Coxiella burnetii by Naturally Infected Hyalomma lusitanicum.

Coxiella burnetii (Derrick) Philip, the causative agent of Q fever, is mainly transmitted by aerosols, but ticks can also be a source of infection. Transstadial and transovarial transmission of C. burnetii by Hyalomma lusitanicum (Koch) has been suggested. There is a close relationship between this tick species, wild animals and C. burnetii but the transmission in a natural environment has not been demonstrated. In this study, we collected 80 engorged nymphs of H. lusitanicum from red deer and wild rabbits. They moult to adults under laboratory conditions and we feed them artificially through silicone membranes after a preconditioning period. C. burnetii DNA was tested in ticks, blood and faeces samples using real-time PCR. The pathogen was found in 36.2% of fed adults, demonstrating that transstadial transmission from nymph to adult occurs in nature. The presence of DNA in the 60.0% of blood samples after artificial feeding confirms that adults transmit the bacteria during feeding. Further studies are needed about co-feeding and other possible transmission routes to define the role of this tick species in the cycle of C. burnetii.

Comparative Ecology of Hyalomma lusitanicum and Hyalomma marginatum Koch, 1844 (Acarina: Ixodidae).

The genus Hyalomma belongs to the Ixodidae family and includes many tick species. Most species in this genus are African species, but two of them, Hyalomma lusitanicum and Hyalomma marginatum, are also found in Europe and, owing to their morphological similarity, it is very difficult to tell them apart. This is a major concern because their phenology and vector capacities are quite different. Moreover, they share many habitats and both are currently spreading to new areas, probably due to climate change and animal/human movements. In this study, we review the described ecology of the two species and provide further interesting data on H. lusitanicum based on the authors' experience, which could be useful in assessing the risk they pose to humans and animals.

Prevalence of Coxiella burnetii (Legionellales: Coxiellaceae) Infection Among Wildlife Species and the Tick Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Ecosystem.

Q fever is a worldwide zoonosis caused by Coxiella burnetii (Derrick) Philip. It is a major cause of abortion among sheep and may be responsible for reproductive losses in red deer in Spain. Airborne transmission is the most widespread; however, some studies suggested that ticks may play a role, but little is known about their actual involvement in the C. burnetii cycle. The aim of this study was to determine the role that Hyalomma lusitanicum (Koch) tick plays in the maintenance of this agent among wildlife in the meso-Mediterranean areas. We processed by PCR 53 swabs from wild rabbits, 21 liver samples from red deer, and 236 ticks collected at different stages. Coxiella burnetii DNA was detected in 43.40% of wild rabbits and 38.09% of red deer, supporting the hypothesis that these animals are quite likely to serve as a reservoir in the field. We also found a high prevalence of C. burnetii in ticks (55.66%). It is worth noting that 50.45% of positive ticks were collected from negative hosts, suggesting that the pathogen probably was acquired at a previous tick stage. Our results suggest transstadial transmission, and the presence of bacterial DNA in the offspring of positive female ticks is the first evidence of the transovarial transmission of C. burnetii by H. lusitanicum. Thus, this tick species seems to play an important role as a bridge of infection in the wildlife cycle, although further studies are needed to confirm vector competence.

Novel Insect Antifeedant and Ixodicidal Nootkatone Derivatives.

Naturally occurring nootkatone, with reported insecticidal and acaricidal properties, has been used as a lead to generate molecular diversity and, consequently, new insect antifeedant and ixodicidal compounds. A total of 22 derivatives were generated by subjecting this molecule to several reactions including dehydrogenation with the iodine/DMSO system, oxidation with SeO2, epoxidation with mCPBA, oxidation or carbon homologations of the α-carbonyl position with TMSOTf (trimethylsilyl trifluoromethanesulfonate) followed by Rubottom and Dess Martin periodane oxidations, condensation with formaldehyde using Yb(OTf)3 as catalyst and dehydroxilation using the Grieco protocol. The insect antifeedant (against Myzus persicae and Ropaloshysum padi) and ixodicidal (against the tick Hyalomma lusitanicum) activities of these compounds were tested. Compound 20 was the most active substance against M. persicae and R. padi, and twice more efficient than nootkatone in the antitick test.

Germacrone Derivatives as new Insecticidal and Acaricidal Compounds: A Structure-Activity Relationship.

Currently, the use of synthetic pesticides is the main method of plant protection applied in agri- and horticulture. However, its excessive use leads to the development of pesticide resistance, a contamination of the environment, toxicity to non-target organisms, and risks for human health. With the ultimate aim of contributing to the develop of a more sustainable pest management, we used the natural product germacrone (compound 1), reported to possess significant insecticidal activity, as starting material for the generation of molecular diversity (2-24). Some of the generated derivatives are natural compounds, such as 1,10-epoxygermacrone (2), 4,5-epoxygermacrone (3), gajutsulactone A (7), germacrol (11), isogermacrone (14), 9-hydroxyeudesma-3,7(11)dien-6-one (19), eudesma-4,7(11),dien-8-one (20), eudesma-3,7(11)-dien-8-one (21) and eudesma-4(15),7(11)-dien-8-one (22). Compounds, 7,11-9,10-diepoxigermacr-4,5-en-8-ol (17), 7,11-epoxieudesma-4,7(11)-dien-8-one (23) and 7,11-epoxieudesma-3,7(11)-dien-8-one (24) are described for the first time. The biocidal activity of most of these compounds was assayed against the tick Hyalomma lusitanicum. The acaricidal effects of compound 24 were four times higher than that of germacrone (1). Compound 2 is an insect antifeedant a thousand times more potent than germacrone against Rhopalosiphum padi, which makes this substance a promising selective antifeedant against this cereal pest.

One-Step Synthesis of Furan Rings from α-Isopropylidene Ketones Mediated by Iodine/DMSO: An Approach to Potent Bioactive Terpenes.

The system I2/dimethyl sulfoxide mediates the one-step transformation of α-isopropylidene ketones into furan rings following a biomimetic approach. This methodology has been used for the synthesis of terpene furans such as mintfurane, curzerene, atractylon, and isoatractylon, all of them possessing interesting biological activities. The synthesis of linderazulene directly from 4,5-epoxygermacrone via a cascade reaction shows the potential of this protocol. Additionally, this compound proved to show significant ixodicidal activity.

Antiparasitic Properties of Cantharidin and the Blister Beetle Berberomeloe majalis (Coleoptera: Meloidae).

Cantharidin (CTD) is a toxic monoterpene produced by blister beetles (Fam. Meloidae) as a chemical defense against predators. Although CTD is highly poisonous to many predator species, some have evolved the ability to feed on poisonous Meloidae, or otherwise beneficially use blister beetles. Great Bustards, Otis tarda, eat CTD-containing Berberomeloe majalis blister beetles, and it has been hypothesized that beetle consumption by these birds reduces parasite load (a case of self-medication). We examined this hypothesis by testing diverse organisms against CTD and extracts of B. majalis hemolymph and bodies. Our results show that all three preparations (CTD and extracts of B. majalis) were toxic to a protozoan (Trichomonas vaginalis), a nematode (Meloidogyne javanica), two insects (Myzus persicae and Rhopalosiphum padi) and a tick (Hyalomma lusitanicum). This not only supports the anti-parasitic hypothesis for beetle consumption, but suggests potential new roles for CTD, under certain conditions.

Spirocercosis in red fox (Vulpes vulpes) in a natural reserve located in a meso-Mediterranean area.

Spirocerca lupi Rudolphi 1809 (Nematoda Spirocercidae) has an indirect cycle involving intermediate and paratenic hosts, having domestic or wild canids as the main definitive hosts. The most frequent pathology affecting dogs is a granulomatous tumor-like growth containing adults in the oesophagus and aorta. There are very few references about the presence of nodules in the stomach. There is scant information about the epidemiology of this disease in wild red foxes in meso-Mediterranean areas. In this work we report on the natural infection of wild red foxes by Spirocerca lupi and the damage produced in the stomach wall in an area where contact with other potential definitive hosts (canids) is very rare. From July 2015 to November 2016, 61 red foxes were sampled. 18.03% of the stomach walls examined had nodules containing Spirocerca lupi adults and eggs. The prevalence of infection was similar for young and adult groups (23.81 and 18.18%; χ2 = 0.25; df = 1; P = 0.6171) and it was higher in females than in males (25.81 versus 10.00%, χ2 = 2.58; df = 1; P = 0.1082). The red fox population in the area seems to be well established judging from their age distribution and the fact that they could be captured all year round. It would appear that in the absence of other main definitive host S. lupi population is able to survive just parasitizing red foxes. Pathologic effects of this parasite on red foxes seems to show some differences related to domestic dogs. These results also indicates as predator control programs can maintain suitable host populations with reduced level of infection.

In vitro feeding of Hyalomma lusitanicum ticks on artificial membranes.

In vitro feeding of ticks (Acari: Ixodidae) is an important means to study the biology of ticks and their vectorial capacity. Here, we have adapted the tick Hyalomma lusitanicum Koch to previously published silicone-based membranes for in vitro feeding. For comparison purposes data on pre-oviposition, oviposition and hatching from females engorged on animals were used. A total of 68 engorged females out of 169 were obtained; feeding duration and feeding behaviour were similar to that of ticks on live host animals, although the final weight achieved for membrane-fed ticks was lower than that of their animal-fed counterparts. Comparison of the time taken for egg production and hatching showed that pre-oviposition was faster for membrane-fed ticks (16 days) than for animal-fed ticks (36 days), whereas the duration of oviposition-hatching was the same for the two feeding methods (34 days). We also observed that seasonality has an influence on tick feeding success: the conditions in Spring/Summer accelerated the tick life cycle. It is concluded that relatively large numbers of homogeneous laboratory-raised Hyalomma ticks can be produced without feeding them on experimental animals.

The effect of excluding ungulates on the abundance of ixodid ticks on wild rabbit (Oryctolagus cuniculus).

Tick abundance depends heavily on host density, so reducing access to the host should reduce tick populations in a determined area. In this study we compared the Parasitic Index (PI = average number of ticks per rabbit) of two wild rabbit populations separated 16 years ago by a fence keeping ungulates from moving freely. Two areas were selected (closed and open) wherein 50 wild rabbits per area were sampled for ticks. The PI in the open area (PI = 989.62) was significantly higher than in the closed area (PI = 515.40). Hyalomma lusitanicum Koch was globally the most abundant species, followed by Rhipicephalus pusillus Gil Collado, Rhipicephalus bursa Canestrini and Fanzago, Haemaphysalis hispanica Gil Collado, Rhipicephalus sanguineus s.l. Latreille and Ixodes ventalloi Gil Collado. Differences between areas varied depending on the species. The number of H. hispanica, R. bursa and R. pusillus were significantly more abundant on rabbits in the closed area, whereas H. lusitanicum predominated in the open area. Ungulates in the open area may have played an important role as the main or alternative host for ticks and/or drawing some tick species away from rabbits. In the closed area other reasons such as inter-specific competition could have influenced the tick abundance. These results show a clear reduction in tick abundance for at least 16 years as well as influenced species distribution.

Seasonal dynamics of ixodid ticks on wild rabbits Oryctolagus cuniculus (Leporidae) from Central Spain.

Rabbit Hemorrhagic Disease and Myxomatosis caused a decline in the rabbit population in the second half of the last century. Despite some recovery, the risk of vector-borne disease is present and thus the importance of controlling vector populations. In the current study, we describe the ixodid tick fauna in wild rabbit in a natural reserve in Ciudad Real (Central Spain) during the course of two 3-year periods (2007-2009 and 2012-2014). Of all the ticks collected on average 72.5 % were larvae, 24.4 % nymphs and 3.1 % adults, although the percentage varied monthly. Seven tick species were identified: Hyalomma lusitanicum Koch (Parasitic indicator [PI] = number of ticks per examined rabbit = 96.47), Rhipicephalus pusillus Gil Collado (PI = 47.37), Haemaphysalis hispanica Gil Collado (PI = 12.15), Ixodes ventalloi Gil Collado (PI = 0.65), R. bursa Canestrini and Fanzago (PI = 0.18), R. sanguineus Latreille (PI = 0.11), Dermacentor marginatus Sulzer (PI = 0.01). In spring and summer, most abundant were larvae of H. lusitanicum, followed by immature stages of R. pusillus and Ha. hispanica. In autumn, the main tick species were nymphs of I. ventalloi whereas in winter adults of Ha. hispanica were more numerous. Rhipicephalus pusillus was present all year long, although not always in high percentage. PI of other species (R. bursa, R. sanguineus and D. marginatus) were too low to be representative. The seasonal dynamics of ticks on wild rabbit defined in this study could be useful to design species-specific control strategies.

Control of Hyalomma lusitanicum (Acari: Ixodidade) Ticks Infesting Oryctolagus cuniculus (Lagomorpha: Leporidae) Using the Entomopathogenic Fungus Beauveria bassiana (Hyocreales: Clavicipitaceae) in Field Conditions.

Entomopathogenic fungi are widely used to control arthropods not just in agricultural settings but also in Veterinary Medicine and Public Health. These products have been employed to control tick populations and tick-borne diseases. The effectiveness of these control measures not only depends on the fungi, but also on the tick species and environmental conditions. In Mesomediterranean areas, tick species are adapted to extreme climatic conditions and it is therefore especially important to develop suitable tick control strategies. The aim of this study was to evaluate the effectiveness of a new method of tick control which entails the application of a commercial strain of Beauveria bassiana (Balsamo, Vuillemin) on wild rabbit burrows under field conditions. Aqueous solutions of the product were applied using a mist blower sprayer into 1,717 burrows. Two trials were performed, one in spring and the other in summer. The parasitic index (PI) was calculated for 10 rabbits per treatment per time point on day +30, +60, and +90 posttreatment and efficiency was calculated by comparing the PI for ticks in treated and untreated rabbits. A total of 20,234 ixodid ticks were collected. Hyalomma lusitanicum Koch, 1844 was the most abundant tick feeding on rabbits. Treatment significantly reduced the PI in spring (by 78.63% and 63.28% on day +30 and +60, respectively; P < 0.05), but appeared to be less effective in summer, with a marginally significant tick reduction of 35.72% on day +30 (P = 0.05). Results suggest that the efficacy of applications inside burrows could be temperature-dependent and that such applications could be an economic alternative to rabbit tick control during at least two months using a diluted solution of B. bassiana conidia.

Francisella species in ticks and animals, Iberian Peninsula.

The presence of Francisella species in 2134 ticks, 93 lagomorphs and 280 small mammals from the Iberian Peninsula was studied. Overall, 19 ticks and 6 lagomorphs were positive for Francisella tularensis subsp. holarctica, suggesting, as described for other regions, that lagomorphs may have an important role in the maintenance of F. tularensis in nature. Of the 6 positive lagomorphs, 4 were identified as the European rabbit, Oryctogalus cuniculus. Additionally, 353 ticks and 3 small mammals were PCR positive for Francisella-like endosymbionts (FLEs) and one small mammal was also positive for Francisella hispaniensis-like DNA sequences. Among FLE positive specimens, a variety of sequence types were detected: ticks were associated with 5 lpnA sequence types, with only one type identified per tick, in contrast to 2 lpnA sequence types detected in a single wood mouse (Apodemus sylvaticus). To our knowledge, this is the first report of FLEs in free-living small mammals as well as the first detection of F. hispaniensis-like sequences in a natural setting.

Long-Term Ecological Study of Host-Seeking Adults of Hyalomma lusitanicum (Acari: Ixodidae) in a Meso-Mediterranean Climate.

From January 2007 to December 2014, three representative meso-Mediterranean bioclimatic environment types were sampled monthly using blanket-dragging techniques to determine the tick abundance rate. Hyalomma lusitanicum Koch, 1844 was the most prevalent species (96.58%) followed by Dermacentor marginatus Sulzer, 1776; Rhipicephalus pusillus Gil Collado, 1936; and Rhipicephalus bursa Canestrini and Fanzago, 1878. H. lusitanicum adults begin questing activity around March, numbers rising quickly reaching their peak in May­June and then diminishing until the end of the year, with a small increase in September­October. This pattern was clear and constant throughout the years, irrespective of the microclimate or biotope tested.

Long term study of ixodid ticks feeding on red deer (Cervus elaphus) in a meso-Mediterranean climate.

Red deer (Cervus elaphus L.) are very valuable in trophy-hunting but also contribute to the preservation of natural areas. They are affected by many parasites and pathogens, including hard ticks that are not only important parasites themselves but can also act as vectors and/or reservoirs of pathogens. Tick phenology is complex insofar as population dynamics depend on environmental conditions, vegetation, host availability and their own intrinsic characteristic. Ticks were collected monthly from January 2007 to December 2014 from red deer on a natural reserve located in a meso-Mediterranean environment in Central Spain. A total of 8978 specimens of ixodid ticks were recovered with a mean Parasitization Index of 65.06 ticks/deer. Red deer were infected the whole year round with a summer-spring pattern and two secondary peaks in February and October. The main species was Hyalomma lusitanicum Koch followed by Rhipicephalus bursa Canestrini and Fanzago, Rhipicephalus pusillus Gil Collado, Dermacentor marginatus Sulzer and Ixodes ricinus L. Hyalomma lusitanicum has a complex life cycle in which several generations initiate their cycle at different times throughout the year, most probably lasting more than 1 year. We also describe the ability of nymphs to feed on large ungulates even though their habitual host is wild rabbit.