**3. Results**

Twenty three of the patients presented at the Burgos Health Services for tick bites were asymptomatic at the time of sampling. Of these patients, 12 were males and 11 were females (Table 1).


Table 1. Characteristics of the patients attended for tick bites in this study

Nine patients were bled once, 13 patients were bled two times and 1 patient was bled in three occasions. All patients were bitten by unknown tick species and in 9 of them the serum samples were withdrawn more than 21 days after the tick bite (Table 2). There was no relation between the tick bite and the age, profession or place where the patient lived.

Of the patients studied, 4 had Abs (IgG) against both *H. lusitanicum* male and female antigens and two of them reacted only with male but not with female whole tick antigen as indicated in Table 2. Two sera showed Abs (IgG) against female but not male *H. lusitanicum* antigens.

Because ticks could have been removed from people in the first 48h post-attachment and even in the case of an effective transmission, the patients could have been bled before the synthesis of the anti-tick Abs have occurred, low values of IgM were found. Afterall, sera from three patients were considered positive against female antigens.

Serum from one patient was positive against male antigens of *Hyalomma lusitanicum.* Two patients showed IgM positive as only response and this fact indicate a possible acute case of *H. lusitanicum* bitten (see in Table 2)*.*

Antibodies to IgE were analyzed in those sera that were positive against both male and female antigens, but we have not observed response to anti-IgE, neither male nor female *H. lusitanicum* antigens.

Relative to the general unexposed population, none of the sera studied were positive against female antigens of *H. lusitanicum* though when sera were assayed against male antigens, one serum was positive (see in Table 3).

anti-IgM (CalBiochem) and 1/500 for anti-IgE (CalBiochem), incubated for 1h at 37ºC. ABTS (2-2´-Azino bis(3-Ethylbenzhia-zoline-6-sulfonic acid) and H2O2 were used as substrates. Reactions were stopped after with 3N sulphuric acid and results were read on a spectrophotometer at 405nm. On each ELISA plate were included negative controls sera obtained from people that never have been bitten by any tick. Experiences were done in triplicate (or more depending on the sera volume). The sample sera were considered positive when their optical density surpased a treshold calculated as the mean optical density of the negative control sera plus three times the standard deviation (mean OD+3δ).

The same parameters were employed when it was referred to the general population.

Twenty three of the patients presented at the Burgos Health Services for tick bites were asymptomatic at the time of sampling. Of these patients, 12 were males and 11 were females

> **Males (%)** 2 (8.69) 4 (17.39) 6 (26.08) **Females (%)** 2 (8.69) 8 (34.78) 1 (4.34)

Nine patients were bled once, 13 patients were bled two times and 1 patient was bled in three occasions. All patients were bitten by unknown tick species and in 9 of them the serum samples were withdrawn more than 21 days after the tick bite (Table 2). There was no relation between the tick bite and the age, profession or place where the patient lived. Of the patients studied, 4 had Abs (IgG) against both *H. lusitanicum* male and female antigens and two of them reacted only with male but not with female whole tick antigen as indicated in Table 2. Two sera showed Abs (IgG) against female but not male *H. lusitanicum*

Because ticks could have been removed from people in the first 48h post-attachment and even in the case of an effective transmission, the patients could have been bled before the synthesis of the anti-tick Abs have occurred, low values of IgM were found. Afterall, sera

Serum from one patient was positive against male antigens of *Hyalomma lusitanicum.* Two patients showed IgM positive as only response and this fact indicate a possible acute case of

Antibodies to IgE were analyzed in those sera that were positive against both male and female antigens, but we have not observed response to anti-IgE, neither male nor female *H.* 

Relative to the general unexposed population, none of the sera studied were positive against female antigens of *H. lusitanicum* though when sera were assayed against male antigens, one

Table 1. Characteristics of the patients attended for tick bites in this study

from three patients were considered positive against female antigens.

**Age-groups (years) 0-10 11-59 60-97** 

**3. Results** 

(Table 1).

antigens.

*H. lusitanicum* bitten (see in Table 2)*.*

serum was positive (see in Table 3).

*lusitanicum* antigens.


Table 2. Reactivity of the human sera from the patients bitten by unspecific ticks, against the *Hyalomma lusitanicum* (male or female) whole antigens.

Screening of the Prevalence of Antibodies to the Tick

and in Spain (Fernández-Soto et al., 2003).

represented in the studied region.

transportation from Spain.

response.

**4. Conclusion** 

by *Hyalomma* ticks.

Asia.

*Hyalomma lusitanicum* in a Province of Northern Spain 193

Burgos is a province of the inner north western region of the Iberian Peninsula. Its climate is continental with cold dry winters and mild summers. Animal husbandry is a very important economical source and livestock parasitism of ticks is common in the region. *Hyalomma* spp. ticks are distributed in Africa, the Mediterranean climatic zone of southern Europe, and in

It is known that ticks are important pets and livestock transmitting tick-borrne diseases. In this sense, though humans are not the preferred hosts of *Hyalomma* ticks and are infrequently bitten in comparison to livestock, sporadic infection of people is usually caused

In fact, Crimea-Congo Haemorrhagic Fever transmitted by *Hyalomma* spp occurs sporadically throughout many areas of Africa, Asia and Europe, but can cause mortality (Estrada-Peña & Jongejan., 1999). Recently a new group of spotted fever has been isolated from *Hyalomma marginatum marginatum* ticks in Morocco (Beati et al., 1997). In Europe have been detected genotipically similar organisms in Portugal (Beati et al., 1995), Croatia (Punda-Polic et al., 2002), Corsica (Matsumoto et al., 2004), Germany (Rumer et al., 2011)

*Hyalomma lusitanicum* is called perinneal specie which is present on cattle year around. These parasitize domestic and wild animals and birds, and are usually abundant in semiarid zones. Its distribution reflects peaks in May-June and October that corresponds to the periods of maximum activity of adults (Habela et al., 1999), but in winter specimens (males and females) remain fixed on their host without feeding (Yousfi-Monod & Aeschliman., 1986). Adult *Hyalomma* actively run out from their resting sites when a host approaches. Cattle, rabbits, hares and deer which are the hosts of *Hyalomma lusitanicum* are well

The frequency with which different tick species bite humans varies significantly from one zone to another and much it depends on the likelihood of humans entering their biotope (human contact with ticks for professional and recreational activities) and the tick affinity for humans. Climatic changes could be probable implicated in the northern establishment of ticks, but perhaps would be more dependent on the introduction of adult females on wild and domestic ruminants due to the uncontrolled movement of livestock as have been proposed recently Rumer et al., (2011) in which the only documented *Hyalomma* spp. tick in Germany was found on a human in the southern part of the country (Lake Constance area) in May 2006, but they did not ruled out the tick

However it is not easy to detect that a person has been bitten by a tick, because people may confuse the bite ticks or other arthropods that might be no elicit antibody

Ticks can inoculate a variety of active molecules during feeding that can block pain, reduce inflammation and suppress or modulate innate and specific acquired immune defences (Brossard & Wikel., 2004). But the duration of the tick attachment may be insufficient to allow for adequate amount of saliva to elicit a detectable antibody response. Sometimes happens that is necessary several tick exposures before the antibody response will be strongly enough to be detected by ELISA. Ticks deposit saliva at the site of their attachment


Table 3. Results of the general population antibodies (IgG) against *Hyalomma lusitanicum*  male and female antigens
