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.

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.

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.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Occurrence of the oribatid mite Trhypochthoniellus longisetus longisetus (Acari: Trhypochthoniidae) on tilapia Oreochromis niloticus.

Mites as parasites infesting fish have been described in a few case reports involving Histiostoma anguillarum, H. papillata, and Schwiebea estradai. We describe the unexpected occurrence of oribatid mites of the genus Trhypochthoniellus on farmed tilapia Oreochromis niloticus. The fish had mites on the skin, fins, and gills, as well as in the mouth. The morphological characteristics of the mites, observed by optical and scanning electron microscopy, were consistent with those described for T. longisetus longisetus. All stages of development were observed, suggesting that the mites were able to actively reproduce on fish.

Tick-borne zoonotic bacteria in ticks collected from central Spain.

The prevalence of tick-borne and related bacteria infecting adult ticks in central Spain was assessed by molecular methods. Six areas were sampled monthly during a 2-year longitudinal study. A total of 1,038 questing and 442 feeding ticks, belonging to eight different species, were tested. The most abundant species were Hyalomma lusitanicum (54% of captures), followed by Dermacentor marginatus (23%) and Rhipicephalus sanguineus (10%). Four human pathogens, including seven Rickettsia species, Anaplasma phagocytophilum, Borrelia burgdorferi, and Francisella tularensis, were detected at percentages of 19.0, 2.2, 1.7, and 0.5, respectively, whereas Bartonella spp. was never detected. In terms of infection and tick abundance, H. lusitanicum seems to be the most significant tick species in the area, carrying three of the five agents tested, and the anthropophilic tick, D. marginatum, infected with Rickettsia spp. and F. tularensis, is the most relevant in terms of public health. The significance of these data is discussed.

Toxicity of oxalic acid against adult Hyalomma lusitanicum ticks (Ixodida: Ixodidae) in laboratory conditions: LD50.

The abundance of Hyalomma lusitanicum ticks in open areas of central Spain is the result of many natural and human factors. Control of tick populations by chemical and other means may be necessary until we can determine the key global factors in this important tick population. Despite many attempts to establish ecological control of ticks, there is little data about the activity of organic acids. This article describes tests on the in vitro toxicity of oxalic acid (OA) against wild H. lusitanicum adult ticks. Serial dilutions of OA dihydrate were prepared in distilled water with 1% of Tween 20. The treatment was applied in three replicates of 10-14 tick/dose from 0 to 1.037 mg OA/tick. Doses > 0.311 mg of OA killed all ticks in 24 h. Toxicity of the OA increased over time; therefore, the LD50 decreased from 0.22 mg at 24 h to 0.127 mg at 72 h. The results obtained show the toxicity of OA against adult H. lusitanicum ticks under in vitro conditions. Comparing toxicity in ticks and honey bees, OA seems a relatively safe treatment for nontarget arthropods and a potential alternative for tick population control. However, further research is needed to establish its real effectiveness and applicability under field conditions.

Azotemia and mortality among Babesia microti-like infected dogs.

Babesia microti-like piroplasms are a recently recognized cause of illness in dogs in northwest Spain. Our objective was to describe the clinical characteristics and investigate the risk factors for azotemia and death among 58 B microti-like infected dogs. Twenty-one of the 58 (36%) dogs were azotemic at the time that the infection was diagnosed. The case fatality rate during the following week was 22%. Dogs with azotemia at the time of diagnosis were 10 times (95% CI, 3.26-28.8) more likely to die during the following week. Azotemia was the main cause of death for B microti-like infected dogs (attributable fraction = 90%). Severe anemia was present in 45 of the 58 (78%) dogs. Azotemic dogs also presented with hyperphosphatemia, hypoalbuminemia, hypercholesterolemia, proteinuria, and high urine protein: creatinine ratios, suggesting a glomerular component to the disease. Age was the only factor significantly associated with the risk of azotemia (P = .042): on average, a 4-year age increase doubled the risk of an infected dog being azotemic. The only factor significantly associated with mortality was azotemia (P = .001). We concluded that B microti-like infection is associated with a high risk of azotemia and mortality.