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.

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.

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.

Control of Tick Infestations in Oryctolagus cuniculus (Lagomorpha: Leporidae) With Spinosad Under Laboratory and Field Conditions.

Because of great economic loss in the world's livestock industry, and the serious risks to human health, the control of ticks and tick-borne diseases is one of the most important health management issues today. Current methodology involves integrated tick control for preventing the development of resistance. Rabbits are hosts for immature stages of the three-host tick Hyalomma lusitanicum Koch; so, we focus on this host as a strategy to interrupt the tick life cycle. Spinosad is an insecticide-acaricide, produced by the fermentation of metabolites of the actinomycete bacterium Saccharopolyspora spinosa. We administered spinosad orally by force-feeding naturally and artificially infested rabbits, and under field conditions by administering treated food via a hopper during the period of peak infestation and reinfestation risk for rabbits. No living larvae were recovered from treated laboratory rabbits. In naturally infested rabbits, the number of live ticks collected from treated rabbits (mean = 0.62 ticks per ear) was significantly lower than those recovered from untreated rabbits (mean = 7.27; P < 0.001), whereas the number of dead ticks collected from untreated rabbits (mean = 6.53) was significantly lower than those recovered from treated rabbits (mean = 18.62; P < 0.001). In addition, free and continually reinfested rabbits freely ingested low doses of spinosad, reducing the tick burden from 48.00 (Day 0) to 26.09 ticks per ear in treated rabbits (Day 16), whereas controls maintained the infection (46.64). This strategy could be useful as an alternative or supplement to traditional acaricides in tick control programs.

Variability of Bartonella genotypes among small mammals in Spain.

In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas-247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)-by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.

Detection of Coxiella burnetii in ticks collected from Central Spain.

A total of 1482 adult ticks collected from vegetation and animals in central Spain in 2003-2005 were tested for the presence of Coxiella burnetii by polymerase chain reaction and subsequent reverse line blot hybridization (PCR-RLB). C. burnetii was identified in 7.7% of questing ticks (80/1039) and 3.4% of ticks collected from animals (15/443) belonging to four species: Hyalomma lusitanicum, Dermacentor marginatus, Rhiphicephalus sanguineus, and R. pusillus. These findings show an active role of ticks in maintaining C. burnetii in wild and peridomestic cycles in central Spain.

Theileria (Babesia) equi and Babesia caballi infections in horses in Galicia, Spain.

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.

Epidemiology of cerebrospinal Elaphostrongylus cervi infection in red deer in central Spain.

Elaphostrongylus cervi produces a subclinical cerebrospinal disease in many wild and domestic ruminants from Europe, North America and New Zealand and has recently been described in Spain. To determine some aspects of its epidemiology, 121 red deer (Cervus elaphus) from central Spain were sampled during 2000. The prevalence (7%) and mean worm burden (3.8 worms per brain) were similar to the values previously recorded in other European areas. The infection was only detected in young deer during the winter. The estimation of larval production in the faeces was not a reliable method of diagnosing E. cervi infection.

Ixodes hexagonus is the main candidate as vector of Theileria annae in northwest Spain.

Babesia canis and Babesia gibsoni have, until recently, been considered the only piroplasms that parasitise dogs. However, recent reports indicate that "small" Babesia infections in Spanish dogs are surprisingly frequent and molecular phylogenetic analysis indicates that the infecting agent is closely related to Babesia microti. Because the 18SrDNA sequence was not completely identical to that of B. microti, the new name "Theileria annae" was assigned to the canine agent. No information is available regarding the possible vector of the new piroplasm, T. annae. As part of an effort to identify the tick that may transmit T. annae in northwest Spain we asked veterinary surgeons practising in the region to collect and send to our laboratory ticks from dogs visiting their clinics. Seven hundred and twenty ticks collected from dogs of unknown clinical status during 1998 and 636 ticks collected between November 2001 and March 2002 from 38 dogs infected with T. annae and 131 uninfected dogs were identified. Results from the first study indicated that among the Ixodidae, Ixodes hexagonus clearly predominates over Ixodes ricinus (26.11% versus 6.67%). This observation was consistent with results of the second study, in which I. hexagonus was detected in all infected dogs and 71.8% of non-infected dogs and I. ricinus was not detected in either the infected or non-infected dogs. Results from the 2001-2002 study also indicate that the presence of Dermacentor reticulatus adult females is significantly less frequent among infected than non-infected dogs (OR=0.44; 95% CI: 0.21-0.92). On the other hand, I. hexagonus adult females and males are 6.75 and 4.24 times more likely to be detected among infected than non-infected dogs, respectively, with the association being, in both cases, statistically significant (95% CI: 1.97-23.12 and 1.92-9.36, respectively). I. hexagonus emerges as the main candidate as vector of T. annae because it feeds on dogs more frequently than other ticks and because B. microti is transmitted by Ixodes ticks, both in North America and Europe. In the absence of definitive confirmation of this hypothesis, our observations suggest that I. hexagonus might serve the same role as does Ixodes scapularis (=Ixodes dammini), the vector of B. microti in eastern North America.

Babesia canis infection in a splenectomized dog.

The present report describes a fatal case of non-experimental babesiosis in a splenctomized 3-year-old fox terrier. A very intense parasitaemia including clusters of up to 16 Babesiae and a prominent haemophagocytic activity were the most relevant findings. A marked leukocytosis, thrombocytopenia and regenerative anaemia were observed. Despite prompt treatment with babesiacidal compounds the condition progressed to acute renal failure and resulted in the death of the animal 48 hours after the onset of symptoms.

Infection of dogs in north-west Spain with a Babesia microti-like agent.

During 1996 a small, ring-shaped, piroplasm was observed in blood smears from 157 dogs in north-west Spain. None of them had previously been in areas endemic for Babesia gibsoni, which was until recently the only small piroplasm known to parasitise dogs. Haematological and serum biochemistry analyses showed that almost all the dogs had an intense regenerative haemolytic anaemia and that in some cases there was evidence of renal failure. A molecular study was made of a sample of the parasite obtained in June 2000. The phylogenetic analysis showed an identity of 100 per cent with the new piroplasm, provisionally denominated as Theileria annae, and 99 per cent with Babesia microti and B. microti-Japan. The results confirm the previous observation of a new form of piroplasm (Theileria annae) which causes disease in dogs in Europe and suggest that it is endemic among the canine population in north-west Spain.

A subtropical case of human babesiosis.

This report constitutes the first well-documented case of symptomatic human babesiosis from a subtropical site, south of the 40th parallel. This paper describes the definitive identification of Babesia divergens infection in a splenectomized patient from the Canary Islands.