Screening of the Prevalence of Antibodies to the Tick Hyalomma lusitanicum in a Province of Northern Spain (Tropical Diseases Due to Protozoa and Helminths) Part 2

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 All patients were bitten by unknown tick species and in 9 of them the serum samples were withdrawn more than 21 days after the tick bite (Table 2).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).

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

Sera (n°) (n=3)	IgG (X±SE)		Sera (n°) (n=3)	IgG (X±SE)
	Females	Males		Females	Males
1	1.036±0.096	0.608±0.015	49	0.650±0.047	0.966±0.058
2	0.897±0.149	0.895±0.048	50	0.585±0.072	0.967±0.036
3	0.465±0.014	1.140±0.053	51	0.276±0.013	1.75±0.025
4	0.377±0.016	0.896±0.015	52	0.296±0.030	0.763±0.031
5	0.887±0.096	0.643±0.100	53	0.479±0.046	0.570±0.053
6	1.031±0.077	0.802±0.055	54	0.346±0.014	0.700±0.176
7	0.279±0.043	0.593±0.088	55	0.299±0.005	0.676±0.022
8	0.915±0.055	0.898±0.038	56	0.817±0.015	0.605±0.127
9	0.630±0.043	0.425±0.045	58	0.343±0.021	1.225±0.067
10	0.544±0.024	0.650±0.088	59	0.415±0.098	0.525±0.037
11	0.378±0.056	0.802±0.065	60	0.349±0.042	0.519±0.123
12	0.311±0.008	0.606±0.060	61	0.434±0.010	0.492±0.035
13	0.447±0.061	0.554±0.025	62	0.430±0.012	1.023±0.008
14	0.117±0.024	0.176±0.009	63	0.325±0.041	0.585±0.103
15	0.386±0.017	0.884±0.060	64	1.040±0.024	1.156±0.193
16	0.778±0.093	0.527±0.103	65	0.824±0.140	0.685±0.015
17	1.019±0.114	0.613±0.038	66	0.424±0.012	0.722±0.094
18	0.938±0.045	0.666±0.030	67	0.498±0.066	1.476±0.055
19	0.234±0.007	0.314±0.017	68	1.077±0.081	1.004±0.082
20	0.485±0.089	0.614±0.055	69	0.927±0.068	0.800±0.172
22	0.671±0.048	0.466±0.045	70	0.234±0.042	0.461±0.107
24	0.473±0.055	0.211±0.026	71	0.323±0.014	0.891±0.200
26	0.383±0.055	0.250±0.058	72	0.876±0.030	0.446±0.130
28	0.389±0.041	0.555±0.082	73	0.524±0.060	ND
29	0.475±0.009	0.167±0.032	74	0.897±0.100	1.182±0.304
30	0.676±0.051	0.306±0.067	76	0.786±0.016	0.516±0.045
31	0.437±0.005	0.810±0.025	77	0.888±0.074	0.922±0.036
32	0.892±0.038	0.206±0.060	79	0.345±0.027	0.856±0.035
33	0.943±0.017	0.148±0.053	80	0.904±0.017	1.408±0.144
34	1.003±0.109	0.957±0.164	81	0.670±0.017	0.826±0.076
35	0.160±0.057	0.499±0.046	82	0.340±0.062	0.866±0.085
36	0.382±0.016	0.943±0.076	83	0.398±0.042	0.985±0.074
37	0.989±0.045	1.093±0.077	84	0.786±0.016	0.578±0.032
38	0.728±0.025	1.400±0.080	85	0.425±0.010	ND
39	0.224±0.055	1.306±0.080	86	0.212±0.007	0.777±0.060
40	0.752±0.067	0.970±0.136	87	0.322±0.061	0.671±0.016
41	0.699±0.018	0.506±0.052	89	1.013±0.117	0.926±0.103
42	0.556±0.019	1.043±0.228	90	0.408±0.091	0.950±0.176
43	0.238±0.013	0.928±0.155	91	0.649±0.062	0.946±0.066
44	0.389±0.029	1.012±0.147	92	0.891±0.016	0.966±0.117
45	0.894±0.084	1.184±0.065	93	0.574±0.084	1.054±0.056
46	0.785±0.089	1.320±0.077	94	0.218±0.057	0.658±0.035
47	0.476±0.011	1.382±0.120	95	0.609±0.055	1.053±0.127
48	0.528±0.033	0.857±0.106	96	0.438±0.055	1.462±0.250
	0.570±0.045				0.780±0.194

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

Conclusion

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 Asia.

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 by Hyalomma ticks.

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

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 represented in the studied region.

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 transportation from Spain.

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 response.

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 to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses but ticks are able to modulate their host’s local haemostatic reactions (Carvalho et al., 2010)

As happens with other ticks in which salivary gland proteins are immunogenic (Sanders et al., 1996), our results have shown that H. lusitanicum has proteins (antigens) that stimulate the production of immunoglobulins in humans as well as the finding of a significant high prevalence in bitten patients by ticks respect to the control group (see in Tables 1 and 2). It would be very interesting to conduct an epidemiological study, in those sera that have positive results, if at any time have had a history of febrile illness of unknown etiology with or without rash.

As occurs with other genus or species of ticks capable to induce high responses, H. lusitanicum could share antigens with other ticks. It is easy to found that antigens can cross react with the antigens of a closely related species. This fact has been observed in several studies that have realized cross-resistance studies between Dermacentor andersoni and Dermacentor variabilis (McTier et al., 1981), Hyalomma anatolicum anatolicum and Boophilus microplus (Parmar et al., 1996), H. a. anatolicum, Hyalomma dromedari and Boophilus microplus (Kumar & Kondal., 1999) as well as between a series of other tick species (Brown & Askenase., 1984; Jaansen van Vuuren et al., 1992). In fact, the homology among several tick species suggests that possibly common antigen(s) may be suitable for a vaccine against some different ticks.

By other hand, though ticks are well known blood suckers, blood sources between males and females seem to be different and gene expressions in fedding males and females are also different (Aljamali et al., 2009). In fact the male blood meals may be digested and nutrients can be used for spermatogenesis.This fact makes that both could be considered as susceptible to bite humans.

Since for each tick-borne disease there may be one or several vectors (Lane, 1994), perhaps this ixodid could be implicated as a vector susceptible of parasitizing humans. In this sense, other studies would in a future provide us about the frequency and which life cycle stages of this tick can infest humans as well as its role in the transmission of human pathogens as happened with those studies in which Dermacentor marginatus was recently demonstrated to be the vector in the transmission of Rickettsia slovaca, that causes the TIBOLA/DEBONEL disease in humans (Rehacek, 1984; Lakos., 1997; Raoult et al., 1997; Ibarra et al., 2006).

Until date, each tick species has preferred environmental conditions and biotopes that determine the geographical distribution of the ticks and the risk areas fro tick borne diseases, but day to day more research studies are going on in order to elucidate a higher diversity of ixodid tick species infesting humans potentially transmisors of underdiagnosed diseases.

In this sense, we consider it would be very interesting to educate primary care physicians in these areas, to be able to identify ticks and how clinicians should deal with patients who have been bitten by ticks, because it would be very interesting to discover potential transmitters of both old and new diseases. It is necessary to develop studies in which ticks removed from the patients must be directly preserved in ethanol and identified because the knowledge about tick species that are susceptible of parasitizing humans is essential for assessing the risks for people who become infected, we can detect pathogens and design measures to prevent infection.