**1. Introduction**

184 Current Topics in Tropical Medicine

Yap, G. S., T. Scharton-Kersten, et al. (1998). "Partially protective vaccination permits the

Yin, G. R., X. L. Meng, et al. (2007). "[Intranasal immunization with mucosal complex

*Immun* 66(9): 4382-4388.

*Ji Sheng Chong Bing Za Zhi* 25(4): 290-294.

development of latency in a normally virulent strain of Toxoplasma gondii." *Infect* 

vaccine protects mice against Toxoplasma gondii]." *Zhongguo Ji Sheng Chong Xue Yu* 

In the recent decades it has been found the occurrence of a large number of hitherto unknown or undervalued pathogens, and they present a risk to health and human welfare. Almost all incidents caused by emerging pathogens have been attached to zoonotic agents, which expanded its host range and are capable of breaching the species barrier (Zeier et al., 2005). Besides the known diseases, new ones emerge or reemerge due to a variety of socioeconomic, health and environmental questions. This increase in communicable diseases has serious implications for public health and animal health (Chomel, 1998; Daszak et al., 2000, Cleaveland et al., 2001, Simpson, 2002; Daszak & Cunningham, 2003; Zeier et al., 2005; Cunningham, 2005; Blancou et al., 2005, Gibbs, 2005; Gortázar et al., 2007).

Emerging zoonoses are also a public health problem, the biggest threat to animal welfare, environmental quality and conservation of biodiversity (Daszak et al., 2000; Cunningham, 2005, Briones et al., 2002). The expected increase contact between humans and wildlife, caused by anthropogenic interference in the ecosystem, increase the emergence of pathogens originating in wildlife cycles, which can cross-infect man and animals (Bengis et al., 2004). Thus, wildlife is a constant source of "zoonotic pool" that plays a fundamental role in human exposure to infectious agents against novel animal (Morse, 1995).

In the recent past, the diseases of wild animals have been important only when they threatened livestock or human health (Daszak et al., 2000), but outbreaks in endangered species have led to them having more significant consideration. Currently, these diseases are booming, especially in the space where interaction occurs between wildlife and farm animals, including an increase in contact between them and the man (Simpson, 2002; Gortázar et al., 2007). Arthropod-borne diseases represent the most common zoonosis in relation to wildlife in the northern hemisphere, especially the Old World (Lindgren et al., 2000), so infectious agents are diverse and constantly growing, so their relationship would be endless (Bueno et al., 2009).

Wild animals and arthropod vectors play also important roles in the exposure of humans and domestic animals to animal-borne pathogens (Morse, 1995). Contact between humans and wild animals may occur when people venture into the latter's ecosystems, such contact in foreseen as a future generator of cross infestation, but the knowledge of ectoparasites, with special mention to ticks and their hosts' reservoirs that are located in many areas of

Screening of the Prevalence of Antibodies to the Tick

Fig. 2. Landscape of the studied area

anthropophilic.

*Hyalomma lusitanicum* in a Province of Northern Spain 187

In this studied area, in the northern sector, the winters are cold and humid and the summers are cold. Vegetation under Atlantic influences consists in oakwood and beechwood, with brushwood. In the southern sector is submediterranean and shows similar winter but hottest summers. Vegetation consists in gall-oak groes and holm-oak wood, being brush scarce (Roman et al., 1996; Dominguez, 2004). Mean summer temperatures in this area range between 16 and 20ºC, while mean winter temperatures range between 2 and 5ºC. Rainfall is usually high in winter at some 900-1100 mm/year. Altitude is ranging between 600-800 meters on the plateau. Climatic differences among different areas of Spain are responsible of

While ticks have a clear geographical distribution in relation to climate, temperature, humidity and like attitude, climate change and global warming have influenced the geographical distribution of ticks. So, some groups as Experts from the International Scientific Working Group (ISW-TBE) on Central European encephalitis transmitted by ticks (Tick-Borne Encephalitis) warn

In Spain, it looks to be *Ixodes ricinus* the most abundant and widespread tick in the Basque Country (Northern Spain) (Barandika et al., 2006) and some authors (Toledo et al., 2009) have observed that one of the most abundant species in Central regions of Spain in terms of infection andtick abundance is *Hyalomma lusitanicum.* However, as happens with other ectoparasites and their host-reservoirs, are located in many areas of Spain that are unstudied or missstudied. In this sense, the genus *Hyalomma* is one of the vectors for *Theileria annulata* that causes Mediterranean theileriosis, and produces considerable economic losses in cattle (Viseras & Garcia Fernandez., 1999) though in terms of public health, this tick is considered as not

Tick bites are generally painless and many people may not even notice the bite and may never find the tick if it falls off. The majority of individuals with tick bites develop no symptoms, and many do not remember getting bitten. The direct damage caused by ticks depends on the number, species and location of the parasites. However, the most harmful effects on animal and public health are derived from indirect vector character (Hillyard, 1996, Wall & Shearer, 1997; Encinas et al., 1999, Sonenshine et al., 2002). Damage can be

of the first detection of these arthropods in areas above 1,500 m above sea level.

both, the diversity of tick species and the circulation of tick-borne pathogens.

world are unstudied. Ticks are vectors of disease have a wide range of pathogens (bacteria, rickettsia, viruses, protozoa and nematodes), affecting both domestic and wild animals, and humans with a zoonotic character. Ticks are considered as one of the most efficient arthropod vector role (Hillyard, 1996, Wall & Shearer, 1997).

In fact, it's known that ticks are vectors of transmission of a number of human viruses that causes Tick-borne meningoencephalitis, Colorado tick fever and Crimean-Congo hemorrhagic fever, among others, bacteria (*Rickettsia* spp, *Anaplasma phagocytophilum*, *Borrelia burgdorferi* and *Francisella turalensis*, among others) (Lopez-Vélez & Molina, 2005; Toledo et al., 2009) and protozoan pathogens including parasites (Lledó et al., 2010) and control of ticks and tick-borne diseases is a major component of animal health programes for the protection of livestock. Over the ixodid tick species which are often found on humans exposed to infested vegetation *Amblyomma* species, *Dermacentor* spp, *Haemaphysalis* spp, *Ixodes* spp and *Hyalomma* spp are found (Estrada- Peña et al., 1999).

In this sense, Genus *Hyalomma* is a phylogenetically young group of ixodid ticks. As proposed Kolonin (2009), domestication and the development of cattle-breeding stimulated the evolution and biological progress of this group. These transformations continue to this day, as is apparent from the great number of intraspecific forms. *Hyalomma* ticks (Figure 1) are medium to large sized, with prominent mouthparts. Most species are 3 hosts, but there are also 1 and 2 hosts. Some species of this genus can use 1, 2 or 3 hosts to develop according to the host they found. The life cycle can last between 3-4 months and more than a year, depending on species and climate. The nymphs and adults stay overwinter in cracks and crevices between the stones of walls and barns, or uncultivated grasslands. Adults are found throughout the year, although the parasite load is higher in spring and summer, parasitizing deer and wild boar. Larvae and nymphs parasitize rabbits and are more prevalent in spring. Though this genus is usually restricted to the Mediterranean region, one of the species, *Hyalomma lusitanicum,* Koch 1844 (*Ixodoidea: Ixodida*) has a widespread distribution in some regions of Southern Spain, from which is introduced by wild animals (Encinas-Grandes, 1986).

Fig. 1. Adult of Hyalomma lusitanicum

This ixodid tick is also located in the Burgos province (north western Spain) (Cordero del Campillo et al., 1994), in areas mainly rural, though recreational activities attracting non-residents have increased in recent years (Figure 2). This tick is not the most prevalent tick in this area.

world are unstudied. Ticks are vectors of disease have a wide range of pathogens (bacteria, rickettsia, viruses, protozoa and nematodes), affecting both domestic and wild animals, and humans with a zoonotic character. Ticks are considered as one of the most efficient

In fact, it's known that ticks are vectors of transmission of a number of human viruses that causes Tick-borne meningoencephalitis, Colorado tick fever and Crimean-Congo hemorrhagic fever, among others, bacteria (*Rickettsia* spp, *Anaplasma phagocytophilum*, *Borrelia burgdorferi* and *Francisella turalensis*, among others) (Lopez-Vélez & Molina, 2005; Toledo et al., 2009) and protozoan pathogens including parasites (Lledó et al., 2010) and control of ticks and tick-borne diseases is a major component of animal health programes for the protection of livestock. Over the ixodid tick species which are often found on humans exposed to infested vegetation *Amblyomma* species, *Dermacentor* spp, *Haemaphysalis* spp,

In this sense, Genus *Hyalomma* is a phylogenetically young group of ixodid ticks. As proposed Kolonin (2009), domestication and the development of cattle-breeding stimulated the evolution and biological progress of this group. These transformations continue to this day, as is apparent from the great number of intraspecific forms. *Hyalomma* ticks (Figure 1) are medium to large sized, with prominent mouthparts. Most species are 3 hosts, but there are also 1 and 2 hosts. Some species of this genus can use 1, 2 or 3 hosts to develop according to the host they found. The life cycle can last between 3-4 months and more than a year, depending on species and climate. The nymphs and adults stay overwinter in cracks and crevices between the stones of walls and barns, or uncultivated grasslands. Adults are found throughout the year, although the parasite load is higher in spring and summer, parasitizing deer and wild boar. Larvae and nymphs parasitize rabbits and are more prevalent in spring. Though this genus is usually restricted to the Mediterranean region, one of the species, *Hyalomma lusitanicum,* Koch 1844 (*Ixodoidea: Ixodida*) has a widespread distribution in some regions of Southern Spain, from which is introduced by

This ixodid tick is also located in the Burgos province (north western Spain) (Cordero del Campillo et al., 1994), in areas mainly rural, though recreational activities attracting non-residents have increased in recent years (Figure 2). This tick is not the most prevalent tick in this area.

arthropod vector role (Hillyard, 1996, Wall & Shearer, 1997).

*Ixodes* spp and *Hyalomma* spp are found (Estrada- Peña et al., 1999).

wild animals (Encinas-Grandes, 1986).

Fig. 1. Adult of Hyalomma lusitanicum

In this studied area, in the northern sector, the winters are cold and humid and the summers are cold. Vegetation under Atlantic influences consists in oakwood and beechwood, with brushwood. In the southern sector is submediterranean and shows similar winter but hottest summers. Vegetation consists in gall-oak groes and holm-oak wood, being brush scarce (Roman et al., 1996; Dominguez, 2004). Mean summer temperatures in this area range between 16 and 20ºC, while mean winter temperatures range between 2 and 5ºC. Rainfall is usually high in winter at some 900-1100 mm/year. Altitude is ranging between 600-800 meters on the plateau. Climatic differences among different areas of Spain are responsible of both, the diversity of tick species and the circulation of tick-borne pathogens.

Fig. 2. Landscape of the studied area

While ticks have a clear geographical distribution in relation to climate, temperature, humidity and like attitude, climate change and global warming have influenced the geographical distribution of ticks. So, some groups as Experts from the International Scientific Working Group (ISW-TBE) on Central European encephalitis transmitted by ticks (Tick-Borne Encephalitis) warn of the first detection of these arthropods in areas above 1,500 m above sea level.

In Spain, it looks to be *Ixodes ricinus* the most abundant and widespread tick in the Basque Country (Northern Spain) (Barandika et al., 2006) and some authors (Toledo et al., 2009) have observed that one of the most abundant species in Central regions of Spain in terms of infection andtick abundance is *Hyalomma lusitanicum.* However, as happens with other ectoparasites and their host-reservoirs, are located in many areas of Spain that are unstudied or missstudied.

In this sense, the genus *Hyalomma* is one of the vectors for *Theileria annulata* that causes Mediterranean theileriosis, and produces considerable economic losses in cattle (Viseras & Garcia Fernandez., 1999) though in terms of public health, this tick is considered as not anthropophilic.

Tick bites are generally painless and many people may not even notice the bite and may never find the tick if it falls off. The majority of individuals with tick bites develop no symptoms, and many do not remember getting bitten. The direct damage caused by ticks depends on the number, species and location of the parasites. However, the most harmful effects on animal and public health are derived from indirect vector character (Hillyard, 1996, Wall & Shearer, 1997; Encinas et al., 1999, Sonenshine et al., 2002). Damage can be

Screening of the Prevalence of Antibodies to the Tick

Fig. 3. Location in the Spain map the Burgos province

**2.2 Human sera** 

revised in 2004.

**2.3 ELISA reactions** 

*Hyalomma lusitanicum* in a Province of Northern Spain 189

The study was carried out on two population groups: sera from people who had been bitten by ticks and serum samples from the general population with no history of tick bites as control group. For the first group, 42 samples of human serum from 23 patients bitten by ticks were randomly collected from patients to the Burgos Health Centres (Figure 2), and the following information was recorded for each person: age, sex, occupation and area of residence. A serum sample was withdrawn from all bitten patients at the time of consultation and the patients were asked to return to provide another sample at 15-20 days later. All residents living in the study area are people related to livestock, or have pets in their care. They are people with an epidemiological history of tick bite, but without identification of the same. For the second group, serum from 97 people was obtained from the general population presented to healthcare centres for reasons unrelated to infectious diseases. All samples were aliquoted and conserved at - 20ºC until use. The survey was performed with the consent of the subjects included in the study, and in compliance with the ethical standards of Alcalá de Henares University´s Comitte on Human experimentation, as well as with the Helsinki Declaration of 1975 as

An indirect ELISA technique was used to detect anti-ticks Abs (IgG, IgM, IgE) in the samples. As antigens were used protein extracts from both males and females of *H. lusitanicum*. ELISA plates were coated with 1µg of antigen per well diluted in 0.1M carbonate/bicarbonate buffer, pH 9.6 and incubated at 4ºC overnight. It was realized three washes in 0.05% PBS-Tween 20. After incubation with 0.05% PBS-Tween 20-1% casein for 1h at 37º and the subsequent washes, 100µl per well of human sera diluted at 1/50 for IgG, 1/20 for IgM and 1/10 for IgE in 0.05% PBS-Tween-20 and incubated for 1h at 37ºC was added. After the subsequent washes, peroxidase-labeled anti-human immunoglobulin antibodies were also diluted in 0.05% PBS-Tween at 1/4000 for both anti-IgG (CalBiochem),

divided, according to the scope of the consequences, cutaneous and systemic. We can point to the inflammatory reaction in the fixation point, which causes itching, scratching, excoriations and self-harm. The reaction can spread awareness of the antigenic components of saliva, causing even anaphylactic shock. The bites often become infected with pyogenic agents such as *Staphylococcus aureus*, not ruling out the occurrence of myiasis (Wall & Shearer, 1997; Encinas et al., 1999, Wall, 2007).Systemic effects include tick paralysis is caused by a neurotoxin secreted by the females of some species, producing a neuromuscular blockade (Sonenshine et al., 2002). *Ixodes ricinus* and *Haemaphysalis punctata* are two species in our area involved in the process of paralysis (Hillyard, 1996, Wall & Shearer, 1997; Encinas et al., 1999). The mechanical transmission of pathogens from sepsis occurs in tick infestation in lambs or calves (Kettle, 1995), not neglecting the effects of blood loss. In this sense, a female has been eating packed up to 4 g of blood, so intense in parasites are common anemias (Encinas et al., 1999).

When ticks bite at time of the attachment they inoculate saliva and occasionally, a neurotoxin secreted at the time of attachment. Saliva in feeding ticks is rich in several biochemical components including various enzymes (Sauer et al., 1995; Giménez-Pardo & Martínez-Grueiro, 2008). Immunogenic and pathogenic proteins enter in the mammalian host during feeding via the tick salivary gland (Kaufman 1989). Their saliva secretions during bites are capable to produce toxicoses and allergic reactions, and in animals it's known that ticks are capable to induce a high humoral immune response (Perez-Sanchez et al., 1992). In human tick attachment are brief and sometimes, are immature forms which introduce a low quantity of antigens which are not enough to induce a good humoral response.

In this paper we i) study the preliminary humoral response in humans, employing antigens from male and female *Hyalomma lusitanicum* tick as a previous work to know if this tick could in a future be capable to bite humans in rural regions being implicated in the transmission of human pathogens and ii) observe differences between males and females in the response that could induce when they bite humans. For this, it was assayed a panel of human sera of different days post bitten by indirect enzyme-linked immunoabsorbent assay technique. This study was carried out in Burgos province (Spain) and results are compared with those obtained from general population and without history of tick bites**.** 

### **2. Material and methods**

#### **2.1 Parasites**

465 unfed adult ticks (males and females) were collected from vegetation in spring and each one was identified by binocular lens. Males (190) were separated from females (275) and processed individually. Specimens were immersed in 70% alcohol for ten minutes rinsed for 30 seconds in Milli-Q water and dried in a filter paper. Each ixodid tick was transferred in PBS-saline (10mM PBS pH 7.2, 146mM NaCl) in a Potter-Elvejem on an ice bath and homogenized. Extracts were removed in eppendorf tubes and centrifuged for 1min at 500 rpm to deposit cuticle fragments and tissue rests. Supernatants were collected and centrifuged in new eppendorf at 14000 rpm for 5 min, filtered through a 0.22µm filter (Millipore) and the resultant one were again filtered through a 0.22µm filter. This whole tick extract was collected and the protein concentration determined using the technique of Bradford (Bradford, 1976) and subsequently adjusted to 1mg/ml for females and 0.635 mg/ml for males using PBS-saline.

#### **2.2 Human sera**

188 Current Topics in Tropical Medicine

divided, according to the scope of the consequences, cutaneous and systemic. We can point to the inflammatory reaction in the fixation point, which causes itching, scratching, excoriations and self-harm. The reaction can spread awareness of the antigenic components of saliva, causing even anaphylactic shock. The bites often become infected with pyogenic agents such as *Staphylococcus aureus*, not ruling out the occurrence of myiasis (Wall & Shearer, 1997; Encinas et al., 1999, Wall, 2007).Systemic effects include tick paralysis is caused by a neurotoxin secreted by the females of some species, producing a neuromuscular blockade (Sonenshine et al., 2002). *Ixodes ricinus* and *Haemaphysalis punctata* are two species in our area involved in the process of paralysis (Hillyard, 1996, Wall & Shearer, 1997; Encinas et al., 1999). The mechanical transmission of pathogens from sepsis occurs in tick infestation in lambs or calves (Kettle, 1995), not neglecting the effects of blood loss. In this sense, a female has been eating packed up to 4 g of blood, so intense in parasites are

When ticks bite at time of the attachment they inoculate saliva and occasionally, a neurotoxin secreted at the time of attachment. Saliva in feeding ticks is rich in several biochemical components including various enzymes (Sauer et al., 1995; Giménez-Pardo & Martínez-Grueiro, 2008). Immunogenic and pathogenic proteins enter in the mammalian host during feeding via the tick salivary gland (Kaufman 1989). Their saliva secretions during bites are capable to produce toxicoses and allergic reactions, and in animals it's known that ticks are capable to induce a high humoral immune response (Perez-Sanchez et al., 1992). In human tick attachment are brief and sometimes, are immature forms which introduce a low quantity of

In this paper we i) study the preliminary humoral response in humans, employing antigens from male and female *Hyalomma lusitanicum* tick as a previous work to know if this tick could in a future be capable to bite humans in rural regions being implicated in the transmission of human pathogens and ii) observe differences between males and females in the response that could induce when they bite humans. For this, it was assayed a panel of human sera of different days post bitten by indirect enzyme-linked immunoabsorbent assay technique. This study was carried out in Burgos province (Spain) and results are compared

465 unfed adult ticks (males and females) were collected from vegetation in spring and each one was identified by binocular lens. Males (190) were separated from females (275) and processed individually. Specimens were immersed in 70% alcohol for ten minutes rinsed for 30 seconds in Milli-Q water and dried in a filter paper. Each ixodid tick was transferred in PBS-saline (10mM PBS pH 7.2, 146mM NaCl) in a Potter-Elvejem on an ice bath and homogenized. Extracts were removed in eppendorf tubes and centrifuged for 1min at 500 rpm to deposit cuticle fragments and tissue rests. Supernatants were collected and centrifuged in new eppendorf at 14000 rpm for 5 min, filtered through a 0.22µm filter (Millipore) and the resultant one were again filtered through a 0.22µm filter. This whole tick extract was collected and the protein concentration determined using the technique of Bradford (Bradford, 1976) and subsequently adjusted to 1mg/ml for females and 0.635

antigens which are not enough to induce a good humoral response.

with those obtained from general population and without history of tick bites**.** 

common anemias (Encinas et al., 1999).

**2. Material and methods** 

mg/ml for males using PBS-saline.

**2.1 Parasites** 

The study was carried out on two population groups: sera from people who had been bitten by ticks and serum samples from the general population with no history of tick bites as control group. For the first group, 42 samples of human serum from 23 patients bitten by ticks were randomly collected from patients to the Burgos Health Centres (Figure 2), and the following information was recorded for each person: age, sex, occupation and area of residence. A serum sample was withdrawn from all bitten patients at the time of consultation and the patients were asked to return to provide another sample at 15-20 days later. All residents living in the study area are people related to livestock, or have pets in their care. They are people with an epidemiological history of tick bite, but without identification of the same. For the second group, serum from 97 people was obtained from the general population presented to healthcare centres for reasons unrelated to infectious diseases. All samples were aliquoted and conserved at - 20ºC until use. The survey was performed with the consent of the subjects included in the study, and in compliance with the ethical standards of Alcalá de Henares University´s Comitte on Human experimentation, as well as with the Helsinki Declaration of 1975 as revised in 2004.

Fig. 3. Location in the Spain map the Burgos province

#### **2.3 ELISA reactions**

An indirect ELISA technique was used to detect anti-ticks Abs (IgG, IgM, IgE) in the samples. As antigens were used protein extracts from both males and females of *H. lusitanicum*. ELISA plates were coated with 1µg of antigen per well diluted in 0.1M carbonate/bicarbonate buffer, pH 9.6 and incubated at 4ºC overnight. It was realized three washes in 0.05% PBS-Tween 20. After incubation with 0.05% PBS-Tween 20-1% casein for 1h at 37º and the subsequent washes, 100µl per well of human sera diluted at 1/50 for IgG, 1/20 for IgM and 1/10 for IgE in 0.05% PBS-Tween-20 and incubated for 1h at 37ºC was added. After the subsequent washes, peroxidase-labeled anti-human immunoglobulin antibodies were also diluted in 0.05% PBS-Tween at 1/4000 for both anti-IgG (CalBiochem),

Screening of the Prevalence of Antibodies to the Tick

 **(p.b)1**

**Patient (nº) Sera (nº) Days**

*Hyalomma lusitanicum* in a Province of Northern Spain 191

**1 5** 1.235±0.046 **2.194±0.144** 0.140±0.014 0.112±0.031 **2 6** 0.856±0.021 0.785±0.074 0.086±0.002 0.163±0.043

**3 8** 1.270±0.094 1.224± 0.161 0.059±0.009 0.148±0.002 **4 9 1.760±0.058 1.903±0.165** 0.102±0.017 0.122±0.028 **5 17a** 17 1.044±0.015 0.928±0.133 0.106±0.009 0.144±0.038

**6 23a** 0.946±0.113 1.199±0.132 **0.399±0.025** 0.023±0.002

**7 36a** 0.724±0.067 0.445±0.095 0.060±0.001 0.050±0.007

**8 80** 1.450±0.050 1.359±0.072 0.068±0.012 0.166±0.015

**9 81** 1.161±0.093 0.674±0.066 0.023±0.004 0.150±0.016

**10 84** 0.926±0.112 0.707±0.166 0.041±0.010 0.173±0.042

**11 189** 1.256±0.187 1.040±0.140 0.100±0.011 0.166±0.041

**12 270** 0.743±0.042 0.714±0.120 0.137±0.002 0.226±0.039

**13 272** 1.289±0.161 1.164±0.038 0.068±0.004 0.060±0.007

**14 279 1.991±0.018 2.139±0.031** 0.150±0.010 0.228±0.017

**15 258 1.730±0.047 1.876±0.205** 0.091±0.009 0.256±0.032

**16 290 1.625±0.095 1.936±0.088** 0.136±0.011 **0.371±0.025** 

**17 293** 1.283±0.033 0.931±0.078 0.029±0.0007 0.055±0.002 **18 303** 0.854±0.073 0.324±0.047 0.089±0.014 0.159±0.009

**19 308** 1.531±0.070 1.104±0.070 0.030±0.012 0.134±0.006

**20 313** 0.974±0.019 0.310±0.026 **0.149±0.010** 0.109±0.015 **21 314** 1.589±0.241 0.512±0.044 0.101±0.014 0.077±0.010

**22 388** 0.624±0.054 0.309±0.037 0.100±0.005 0.111±0.009

**23 354** 1.072±0.239 1.096±0.026 0.121±0.014 0.195±0.014

**Control sera (n=8)** 0.996±0.149 0.782±0.098 0.086±0.013 0.149±0.021 Table 2. Reactivity of the human sera from the patients bitten by unspecific ticks, against the

*Hyalomma lusitanicum* (male or female) whole antigens.

**7** 0.975±0.077 1.098±0.207 0.079±0.022 0.165±0.022

**17b** 0.816±0.040 0.499±0.062 0.100±0.026 0.057±0.027

**23b** 25 1.291±0.201 1.268±0.111 **0.350±0.033** 0.144±0.046

**36b** 11 1.442±0.124 1.229±0.024 0.046±0.010 0.134±0.019 **50** 20 1.033±0.061 0.516±0.036 0.050±0.012 0.121±0.021

**79** 1.443±0.190 1.250±0.138 0.107±0.004 0.187±0.005

**82** 0.404±0.042 0 0.062±0.004 0.175±0.031

**83** 1.241±0.011 0 0.040±0.004 0.171±0.039

**190** 22 1.378±0.184 1.136±0.031 0.099±0.004 0.166±0.040

**271** 33 0.872±0.009 0.626±0.176 0.146±0.016 0.238±0.025

**273** 25 1.057±0.074 0.766±0.072 0.077±0.010 0.097±0.009

**280** 30 **2.152±0.029 1.767±0.029** 0.119±0.009 0.290±0.032

**259** 13 **1.750±0.051 1.496±0.150** 0.107±0.007 0.259±0.045

**291** 27 **1.628±0.070 1.864±0.101** 0.114±0.007 **0.316±0.020** 

**304** 18 0.883±0.074 0.517±0.046 0.055±0.007 0.156±0.018

**309** 20 1.309±0.098 0.833±0.045 0.054±0.008 0.110±0.025

**315** 24 **1.891±0.055** 0.493±0.007 0.120±0.010 0.116±0.026

**389** 22 0.664±0.070 0.088±0.026 0.063±0.007 0.127±0.021

**355** 25 **1.711±0.054** 0.251±0.077 **0.237±0.028** 0.214±0.009

**IgG (Χ±SE) IgM (Χ±SE) Female (Ag) Males(Ag) Females(Ag) Males(Ag)** 

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.
