Parasitology Research

, Volume 109, Issue 1, pp 147–162

Ticks’ response to feeding on host immunized with glandular extracts of Rhipicephalus sanguineus females fed for 2, 4, and 6 days. I. Inactivity or early degeneration of salivary glands?

Authors

  • Karim Christina Scopinho Furquim
    • Departamento de Patologia VeterináriaFCAV, UNESP
  • Maria Izabel Camargo Mathias
    • Departamento de BiologiaInstituto de Biociências, UNESP
  • Letícia Maria Gráballos Ferraz Hebling
    • Departamento de BiologiaInstituto de Biociências, UNESP
  • Gislaine Cristina Roma
    • Departamento de BiologiaInstituto de Biociências, UNESP
    • Departamento de Patologia VeterináriaFCAV, UNESP
Original Paper

DOI: 10.1007/s00436-010-2238-7

Cite this article as:
Furquim, K.C.S., Mathias, M.I.C., Hebling, L.M.G.F. et al. Parasitol Res (2011) 109: 147. doi:10.1007/s00436-010-2238-7

Abstract

The present study histologically analyzed the salivary glands of Rhipicephalus sanguineus females fed for 2, 4, and 6 days in hosts which had been previously immunized with glandular extracts obtained from females from this same species in different periods of feeding, having as main objective verify the action of these extracts in the secretor cycle of these glands. For this, glandular extract of females fed for 2 days (SGE2), glandular extract of females fed for 4 days (SGE4), and glandular extract of females fed for 6 days (SGE6) extracts were obtained from salivary glands of R. sanguineus females fed for 2, 4, and 6 days respectively. Then, New Zealand White naive rabbits were inoculated either with extracts (test group = TG), or with a mixture of phosphate buffer and Freund's complete adjuvant (control group 2 = CG2). Each inoculated rabbit (TG and CG2) and non-inoculated (control group 1 = CG1) was posteriorly infested with 15 couples of fasting R. sanguineus from which the salivary glands had been collected from females fed for 2, 4, and 6 days. The results revealed that the resistance the hosts had acquired by the immunization with the extracts affected differently the secretory activity of the glandular cells. It was verified that the resistance to SGE2 and SGE4 extracts acted in the cells of acini II and III, being c1 and c5 from II and d from III inactivated due to the action of SGE2 and c1 and c4 from II and f from III inactivated by the action of SGE4. As for the resistance to SGE6 the effect was only on cells of acini II (c1, c3 e c4), which were also inactivated. In addition, the hosts’ resistance to SGE2SGE6 extracts made the degenerative process earlier in comparison to CG1. On the other hand, the resistance to the extracts did not influence the characteristics of the degenerative process normally found in salivary glands. The assynchronism of the degenerative process was maintained—acini III were always the most affected and I the less affected. The structural cell alterations, such as cytoplasmic vacuolation, nuclear alterations and formation of apoptotic bodies which characterize the occurrence of atypical apoptosis were also maintained in the glands of individuals from TG making it clear that the immunization of the hosts with glandular extracts SGE2–SGE6 had influenced the glandular physiology of R. sanguineus, which is an important piece of information in the search for a way to control these ectoparasites.

Introduction

The pharmacological and immunological properties of ticks’ saliva provide them with a very successful spoliative action on different species of animals (Ribeiro et al. 1985; Wikel 1999). The saliva is a complex and efficient mixture which acts in the modulation of immune-inflammatory modulation of the host, allowing feeding and the permanence on the host (Ribeiro et al. 1985; Wikel 1999; Sauer et al. 2000). In addition, the fact that the saliva immunosuppresses the host makes the ticks distinguished transmitters of pathogenic agents which are veiculated by the saliva itself (Wikel 1999).

The ticks’ saliva is produced by salivary glands, organs that present great morphofunctional complexity (Till 1961; Binnington 1978; Balashov 1983; Walker et al. 1985; Fawcett et al. 1986; Gill and Walker 1987; Sonenshine 1991; Marzouk and Darwish 1994). In the females they are composed by types I, II, and III acini (Binnington 1978; Walker et al. 1985; Gill and Walker 1987; Sonenshine 1991). Considering the importance of acini II and III for the feeding and attachment of the ticks (Binnington 1978; Walker et al. 1985), as well as in the release of pathogens, these have to be deeply studied (Jittapalapong et al. 2008).

In the ticks, acini II are constituted by a, b, c1, c2, c3 e c4 cells (Binnington 1978) and in Rhipicephalus sanguineus females c5 and c6 (Furquim et al., submitted paper) were additionally found. It is known that a cells are involved with cement secretion for the construction of the fixation cone (Binnington 1978; Walker et al. 1985; Fawcett et al. 1986; Gill and Walker 1987), and b and c with several functions that have been attributed to the saliva in the modulation of the host’s immune-inflammatory response (Binnington 1978; Walker et al. 1985). Acini III are formed by d, e and f cells (Binnington 1978; Walker et al. 1985; Gill and Walker 1987). d and e secrete components of the cement (Binnington 1978; Walker et al. 1985; Gill and Walker 1987) and f have two functions: secretory and osmoregulative (Binnington 1978; Walker et al. 1985; Gill and Walker 1987).

In the ticks, the salivary glands (Wikel et al. 1978; Gill et al. 1986; Almeida et al. 1994; Ferreira et al. 1996; Szabó and Bechara 1997; Jittapalapong et al. 2000a; Nunes et al. 2010) and the intestine (Szabó and Bechara 1997; Jittapalapong et al. 2000a; Caperucci et al. 2009, 2010; Veronez et al. 2010) are important sites of antigens production. Because of this, after the first contact of these ectoparasites with some hosts, the latter develop resistance (Wikel et al. 1978; Gill et al. 1986; Jittapalapong et al. 2000a; Zhou et al. 2006). In this sense, several studies are carried out to verify the acquisition of resistance by the hosts when these are immunized by successive infestations (Nunes et al. 2010; Monteiro and Bechara 2008; Monteiro et al. 2010; Veronez et al. 2010; Caperucci et al. 2009, 2010; Jittapalapong et al. 2000a, b) or by the inoculation of extracts of whole ticks or part of them (Wikel 1981; Ferreira et al. 1996; Szabó and Bechara 1997; Jittapalapong et al. 2000a, b, 2008).

The acquisition of resistance by the hosts is measured by the analysis of feeding and reproductive parameters of infesting ticks of previously immunized animals (Wikel 1981; Szabó and Bechara 1997; Jittapalapong et al. 2000b), as well as by the analysis of the impact of this resistance in the salivary glands (Sanders et al. 1996; Jittapalapong et al. 2008; Nunes et al. 2010) and intestines (Veronez et al. 2010; Caperucci et al. 2009, 2010) of different species.

According to Wikel (1981) and Jittapalapong et al. (2000b, 2008) the immunization from antigens originated in the salivary glands would stimulate the immunological response of the hosts which would affect the ticks of subsequent infestations. This would be caused by the direct action of the resistance acquired by the host in the salivary glands’ secretory cycle reducing the efficiency of the feeding process and the transmission of pathogens by the ticks (Jittapalapong et al. 2008).

There are no studies which address the immunization of hosts with extracts of salivary glands of R. sanguineus females in different periods of the glandular cycle, which would provide important information to understand how the host’s organism would respond to pre-immunization with the different glandular components of this species. The interest in studies of this nature would be justified by the fact that R. sanguineus species is considered cosmopolite and the most predominant of Ixodidae family (Pegram et al. 1987a, b). In addition to being an important transmitter of diseases like botounneuse fever for the men and babesiosis and erchilliosis for dogs (Walker et al. 2000). Furthermore, the ability of R. sanguineus to transmit Rickettsia rickettsii—the causing agent of Rocky Mountain spotted fever to the human being (Demma et al. 2005) and canine visceral leishmaniosis (Coutinho et al. 2005; Dantas-Torres 2008).

Considering the information above, the present study had the objective to histologically analyze the salivary glands of R. sanguineus females with 2, 4, and 6 days of feeding subjected to the infestation on hosts previously immunized with glandular extracts.

Material and methods

Material

To perform this study, salivary glands of adult R. sanguineus females fed for 2, 4, and 6 days subjected to infestation on New Zealand White rabbits immunized with glandular extracts were used. For this, fasting individuals (males and females) from a colony kept in BOD incubator, in controlled conditions (29°C, 80% of humidity and photoperiod of 12 h), in the Biotery of the Biology Department of UNESP campus Rio Claro, SP were used in the infestations A, B and C made in rabbits according to the procedure described by Bechara et al. (1995).
  • Infestation A: made in naive rabbits using 25 couples of R. sanguineus couples/host, for the acquisition of females of ticks fed for 2 (55 individuals), 4 (36 individuals), and 6 (25 individuals) days, for the obtainance of glandular extracts: SGE2 = glandular extract of females fed for 2 days, SGE4 = 4 days, and SGE6 = 6 days.

The chosen periods of feeding (2, 4, and 6 days of feeding) were determined based on the cycle of salivary gland secretion, once fasting individuals have glands containing a, c1, c3, d, and e active; those fed for 2 days presented activation of b, c2, c4, c5, c6, and f cells, in those fed for 4 days cells c5 and f became inactive remaining alive only cells a, b, c1c4, and c6 and finally individuals fed for 6 days had inactivation of c6 cells (Furquim et al., submitted paper).

The extracts were processed and inoculated in the hosts subjected to infestation B.
  • Infestation B (test group = TG): was made in 12 sensitized rabbits, four of them inoculated with SGE2 extract, four with SGE4, and four with SGE6, which were subjected to challenge infestation with 15 couples of adult R. sanguineus ticks/host.

  • Infestation C (control group = CG): made in eight naive rabbits, considering that four of these animals were not inoculated (control group 1 = CG1) and four were inoculated with a mixture of the Freund’s complete adjuvant and phosphate buffer (control group 2 = CG2) and then these animals were subjected to challenge infestation with 15 couples of adult R. sanguineus ticks/host.

This experiment was approved by the Ethics Committee in Research and Scientific Merit–UNIARARAS, Protocol n° 021/2009.

Methods

In the Laboratory of the Department of Molecular Biology of UNESP Rio Claro, SP, Brazil, the salivary glands were put separately (glands of females fed for 2, 4, and 6 days) in Eppendorfs tubes containing 200 μL of phosphate buffer pH 7.4. They were then macerated, centrifuged for 30 min at 10,000×g, the supernatant was collected and was put for proteins dosage, which occurred according to the methodology described by Sedmak and Grossberg (1977) (Bradford method), which should be of at least 0.2 μg/μL.

After the determination of the protein content of each sample, the extracts were filtered, separately by situation of study, with the help of sterile filtering units (JBR610303, disposable filtering unit Millex GV, durapore membrane PVDF, Millipore, MilliUni), of 0.22 μm and 13 mm of diameter, attached to hypodermic syringes in the interior of a pre-sterile vertical laminar flow. The extracts were then divided in volumes of 50 μL and kept in freezer at −20°C. Only at the moment of inoculation the extracts were mixed (50 μL of extract/host) with 50 μL of complete Freund’s complete adjuvant (reference # F 5881, Sigma-Aldrich), as well as the mixture of Freund’s complete adjuvant with phosphate buffer pH 7.4 (FA + PBS), procedures that are also made in pre-sterile vertical laminar flow.

After, rabbits from TG and CG2 had the right dorsal side sheared and were subcutaneously inoculated with SGE2, SGE4, and SGE6 extracts, as well as with FA + PBS, via hypodermic syringe, for three times in intervals of 21 days. Only after 15 days from the last inoculation, all the hosts from TG and CG were subjected to infestation challenge with 15 couples of R. sanguineus/host.

In the sequence, R. sanguineus females fed for 2, 4, and 6 days were removed from the rabbits inoculated with SGE2, SGE4 e SGE6 (GT) extracts, with the mixture FA + PBS (GC2), as well as from those not inoculated (CGI) and subjected to histological analysis.

Histological analysis

In the Histology Laboratory of the Biology Department of UNESP campus Rio Claro, SP, Brazil, salivary glands of R. sanguineus females of each group (TG e CG1 e 2) fed for 2, 4, and 6 days were removed in buffered saline solution (7.5 g NaCl + 2.38 g Na2HPO4 + 2.72 g KH2PO4 in 1,000 mL distilled water) and fixed in 4% paraformaldehyde at 4°C. After fixation, the material was dehydrated in a series of increasing concentrations of ethanol (70%, 80%, 90%, and 95%), embedded in resin (Leica), and sectioned at 3 μm thickness. Sections were mounted on glass slides and stained with hematoxylin-eosin. Slides were mounted with Canada balsam and examined and photographed under Motic BA 300 light microscope.

Results

Control group 1

  • Females fed for 2 days
    • Salivary glands are intact (Fig. 1a–c).

    • Acini I present dilated lumen (Fig. 1a).

    • Acini II present reduced lumen and contain cells a, c1, c2, c3, c4 and c5 all of them full of secretion (Fig. 1a–b).

    • Acini III present reduced lumen and contain cells d, e, and f, all of them full of secretion (Fig. 1c).

    https://static-content.springer.com/image/art%3A10.1007%2Fs00436-010-2238-7/MediaObjects/436_2010_2238_Fig1_HTML.gif
    Fig. 1

    Histological sections of salivary glands of Rhipicephalus sanguineus females used as control group 1 (CG1). a–c Two days of feeding presenting the glandular tissue intact, synthesis activity and secretion and cells onset a (a), c1, c2, c3, c4, and c5 of acini II (II) (a–b) and d, e, and f of III (III) (c) full of secretion. d–g Four days of feeding with intact glands and intense secretory activity. In acini II (II) there are cells a, c2, c3, c4, and c5 (e, f) and in III cells d (d), e and f (III) (d, g), the latter without secretion. Acini II (II) present dilated lumen (lu) (e–f). Acini III (III) present reduced (d) or dilated (g) lumen (lu). h–n Six days of feeding with acini II (II) (j), III (III) (k–l) and indeterminate (Ind) (m) presenting signs of degeneration. Acini II (II) are intact or in degeneration (j). Acini III (III) present reduced (k) or dilated (l) lumen (lu) and their cells f (f) (k–l) present intense cytoplasmic vacuolation (va). Acini in degeneration present loss of cellular contact (arrow) (j–k), cytoplasmic vacuolation (va) (j–m), irregular (in) (j, k, m), picnotic (pn) (l), and fragmented (dotted rectangle) (k) nuclei. There are also apoptotic bodies (ab) (n). I acinus type I, lu acinus lumen, n nucleus; a cell a, c1 cell c1, c2 cell c2, c3 cell c3, c4 cell c4, d cell d, e cell e. Bars 50 μm

  • Females fed for 4 days
    • Salivary glands are intact (Fig. 1d–g).

    • Acini I with reduced (Fig. 1d) or dilated (Fig. 1e) lumen.

    • Acini II present slight increase in the diameter of the lumen (Fig. 1e–f) and cells a, c2, c3, c4, and c5 are observed, all of them full of secretion granules (Fig. 1e–f).

    • Acini III present reduced lumen (Fig. 1d) or a slight increase in the diameter (Fig. 1g), in addition, cells d, e and f are also observed, d and e full of secretion granules (Fig. d, g).

  • Females fed for 6 days
    • In these, acini I, II, and III and Indeterminate are present, the latter scarce and this way named due to the advanced stage of degeneration which made their identification difficult and countless apoptotic bodies (Fig. 1h–n). Acini II and III also present degenerative characteristics, more intense in the latter (Fig. 1j–l).

    • Acini I present very dilated lumen (Fig.1h).

    • Acini II present reduced lumen, intact (Fig. 1i) or in degeneration (beginning of vacuolation; Fig. 1j), the latter being more numerous. Secretory cells a, c1, c2, c3, and c4 are also observed, full of secretion granules (Fig. 1i–j).

    • As for acini III, these can present reduced (Fig. 1k) or dilated (Fig. 1l) lumen. In addition, they present d, e, and f cells, only d and e contain secretion granules and f, cytoplasmic vacuolation (Fig. 1k–l).

Control group

The salivary glands of females fed for 2, 4, and 6 days on the hosts of this group presented similar characteristics to the females from CG1 fed for 2, 4, and 6 days, respectively.

Test group

Inoculation of glandular extract of 2 days

  • Females fed for 2 days
    • These salivary glands were intact (Fig. 2a–e).

    • Acini I presented reduced (Fig. 2a) or dilated (Fig. 2b) lumen.

    • Acini II presented reduced lumen (Fig. 2b–d). Cells a, c2, c3, c4 and c5 are present, all of them full of secretion (Fig. 2b–d).

    • Acini III present reduced lumen and in them there are d, e, and f cells, all of them full of secretion (Fig. 2e).

    https://static-content.springer.com/image/art%3A10.1007%2Fs00436-010-2238-7/MediaObjects/436_2010_2238_Fig2_HTML.gif
    Fig. 2

    Histological sections of salivary glands of Rhipicephalus sanguineus females used as test group TG (SGE2). a–e Two days of feeding with intact glandular tissue, in the beginning of synthesis activity an secretion with the cells aa, c2, c3, c4, and c5 of acini II (II) (b–d) and d (d), e (e), and f (f) of acini III (III) (e) full of secretion. f–k Four days of feeding with acini II (II) (h) and III (III) (i–j) presenting degeneration signs. Acini III (III) present reduced (i) or dilated (j) lumen (lu) and their cells f (f) present intense cytoplasmic vacuolation (va). Acini in degeneration present loss of cellular contact (arrow) (h), cytoplasmic vacuolation (va) (h–i), irregular (in) (h–j) and picnotic (pn) (h–i) nuclei. There are also apoptotic bodies (ab) (k). l–q Six days of feeding with acini I (l), II (m), III (n–o) and indeterminate (Ind) (p) in degeneration. Acini III present reduced (n) or dilated (o) lumen (lu) and their cells f (f) present intense cytoplasmic vacuolation (va). Acini in degeneration present loss of cellular contact (arrow) (m), cytoplasmic vacuolation (va) (m–p), irregular nuclei (in) (o–p), picnotic nuclei (pn) (l–n) and nuclei with chromatic marginalization (dotted circle) (m). There are also apoptotic bodies (ab) (q). I acinus type I, dt duct, n nucleus, v valve, lu acinus lumen, asterisk cell without secretion granules, a cell a, c2 cell c2, c3 cell c3, c4 cell c4, d cell d, e cell e, f cell f. Bars 50 μm

  • Females fed for 4 days
    • These glands present signs of degeneration such as beginning of cytoplasmic vacuolation (Fig. 2h–j) and the presence of apoptotic bodies (Fig. 2k).

    • Acini I are intact and can present reduced (Fig. 2f) or dilated (Fig. 2g) lumen.

    • Acini II are starting to degenerate, present increase size in relation to those from females fed for 2 days and subjected to the extract of 2 days and reduced lumen (Fig. 2h). In addition, many acini II present cells without secretion granules (Fig. 2h).

    • In acini II cells a, c2, c3, and c4 are observed, all of them full of secretion granules (Fig. 2h).

    • Acini III are in process of degeneration and can present reduced (Fig. 2i) or slightly dilated (Fig. 2j) lumen, being the former more numerous.

    • In acini III cells d, e, and f are found (Fig. 2i–j). Cells d and e are full of secretion granules and in f, the cytoplasm does not contain secretion granules in addition to being disorganized due to the beginning of vacuolation(Fig. 2i–j).

  • Females fed for 6 days
    • Glands are in degeneration (Fig. 2l–p), beginning cytoplasmic vauolation (Fig. 2m–p), which is more intense than that observed in the females fed for 4 days and subjected to glandular extract of days as well as the presence of apoptotic bodies (Fig. 2q).

    • All kinds of acini with degenerative characteristics are found (Fig. 2l–p).

    • Acini I are in degeneration and present very dilated lumen (Fig. 2l).

    • Acini II are in degeneration, with reduced lumen and with cells a, c2, c3, and c4, all full of secretion granules (Fig. 2m). In addition, some acini with cells free of secretion granules were found (Fig. 2m).

    • Acini III are in degeneration, therefore presenting irregular shape, cytoplasmic vacuolation, with reduced (Fig. 2n) or dilated (Fig. 2o) lumen, being the latter more numerous.

    • In acini III only cells e are found, full of secretion granules, and f, free of secretion and presenting cytoplasmic vacuolation (Fig. 2n–o).

Inoculation of glandular extract of 4 days

  • Females fed for 2 days
    • Glands are intact (Fig. 3a–f).

    • Acini I present dilated lumen and with irregular shape (Fig. 3a).

    • Acini II present reduced (more numerous) lumen (Fig. 3b) or slight increase in the diameter (Fig. 3c–d). Cells a, c2, c3, c4, and c5, contain secretion (Fig. 3b–d).

    • Acini III present reduced (Fig. 3e) or dilated lumen (Fig. 3f), being the former more numerous. Cells d, e and f are all full of secretion (Fig. 3e–f).

    https://static-content.springer.com/image/art%3A10.1007%2Fs00436-010-2238-7/MediaObjects/436_2010_2238_Fig3_HTML.gif
    Fig. 3

    Histological sections of the salivary glands of Rhipicephalus sanguineus femalesused as test group TG (SGE4). a–f Two days of feeding with intact cells, in the beginning of the secretory activity and cells a, c2, c3, c4, and c5 of acini II (II) (b–d) and d (d), e (e), e f (f) of acini III (III) (e–f) full of secretion. Acini II (II) and III (III) present reduced (b, e) or dilated (c–d, f) lumen (lu). g–m Four days of feeding with degenerative signs in acini II (II) (h–j) and III (III) (k–l). Acini II (II) and III (III) present reduced (h, j–k) or dilated (i, l) lumen (lu). Acini in degeneration present loss of cellular contact (arrow) (h, j), cytoplasmic vacuolation (va) (h–i, k–l), irregular nuclei (in) (h, k–l), picnotic nuclei (pn) (h–i) and nuclei with chromatic marginalization (dotted circle) (i–j). There are also apoptotic bodies (ab) (m). n–r Six days of feeding with acini I (I) (n), II (II) (o), III (III) (p) and indeterminate (Ind) (q) in degeneration. In acini III (III) the cells f (f) present intense cytoplasmic vacuolation (va). Acini in degeneration present loss of cellular contact (arrow) (o), cytoplasmic vacuolation (va) (n–q), irregular (in) (n–p), picnotic (dotted circle) (n–p) and fragmented (dotted rectangle) (p) nuclei. Apoptotic bodies (ab) (r) are also observed. I acinus type I, lu acinus lumen, n nucleus, v valve, dt duct, a cell a, c1 cell c1, c2 cell c2, c3 cell c3, c4 cell c4, c5 cell c5, d cell d, e cell e, f cell f. Bars 50 μm

  • Females fed for 4 days
    • Degenerative signs are found in acini II (Fig. 3h–j) and III (Fig. 3k–j) being more numerous in the latter. In addition, apoptotic bodies are also found (Fig. 3m).

    • Acini I are intact and present dilated lumen (Fig. 3g).

    • Acini II are little developed, starting cytoplasmic vacuolation (Fig. 3h–i) and some with dilated lumen (Fig. 3i), while others present reduced lumen (Fig. 3h, j). Cells a, c1, c2, c3, e c5 are found (Fig. 3h–j).

    • Acini III present reduced (Fig. 3k) or dilated lumen (Fig. 3l), the latter more numerous.

    • In acini III cells d, e (Fig. 3k–l), and f (Fig. 3k) are present, the latter presenting degenerative signs, such as cytoplasmic vacuolation, more significant.

  • Females fed for 6 days
    • These glands present degenerative signs such as vacuolation of glandular cells (Fig. 3n–q), nuclear fragmentation (Fig. 3p) and presence of apoptotic bodies (Fig. 3r), signs that are more intense than those detected in the salivary glands of females fed for 4 days subjected to glandular extract of 4 days.

    • In these glands acini I, II, and III were found (Fig. 3n–p), with degenerative characteristics such as cytoplasmic vacuolation and nuclear fragmentation. In addition, acini in advanced degenerative stage were found, and as they could not be identified were named indeterminate (Fig. 3q).

    • Acini I present reduced or dilated lumen (Fig. 3n).

    • Acini II present degeneration signs, reduced lumen and cells a, c1, c2, c3, and c4, contain secretion (Fig. 3o).

    • All acini III present degenerative characteristics and reduced lumen. In addition, cells d and e are full of secretion granules (Fig. 3p).

Inoculation of glandular extract of 6 days

  • Females fed for 2 days
    • Salivary glands are intact (Fig. 4a–d).

    • Acini I present reduced or dilated lumen (Fig. 4a), the former more numerous and irregular-shaped.

    • Acini II present reduced lumen and cells a, c2, c3, c4, and c5 are full of secretion (Fig. 4b–c).

    • Acini III present reduced lumen and cells d, e and f, contain secretion (Fig. 4d).

    https://static-content.springer.com/image/art%3A10.1007%2Fs00436-010-2238-7/MediaObjects/436_2010_2238_Fig4_HTML.gif
    Fig. 4

    Histological sections of salivary glands of Rhipicephalus sanguineus females used as test group TG (SGE6). a–d Two days of feeding with intact glandular tissue, beginning of the secretion phase with cells a, c2, c3, c4, and c5 dos acini II (II) (b–c) e d (d), e (e) e f (f) (d) of III (III) full of secretion. e–k Four days of feeding with intact (I) acini I (e) or in degeneration (f) and acini II (II) and III (III) in degeneration (g–j). Os acini III (III) present reduced (i) or dilated (j) lumen (lu). In acini in process of degeneration are observed loss of cellular contact (arrow) (h–j), cytoplasmic vacuolation (va) (f, i–j), irregular (in) (e, g, i–j), and picnotic (pn) (g) nuclei. Apoptotic bodies are also observed (ab) (k). l–q Six days of feeding with acini I (i) (l), II (II) (m–n), III (III) (o) and indeterminate (Ind) (p) in degeneration. In acini III (III) the cells f (f) (o) present intense cytoplasmic vacuolation (va). Acini in degeneration present loss of cellular contact (arrow) (m–n), cytoplasmic vacuolation (va) (m–o), irregular (in) (m, o–p) and picnotic (pn) (l, o) nuclei, with chromatic marginalization (dotted circle) (n, p) and fragmented nuclei (dotted rectangle) (o). Apoptotic bodies are also observed (ab) (q). I type I acinus, lu acinus lumen, n nucleus, dt duct, v valve, a cell a, c1 cell c1, c2 cell c2, c3 cell c3, c4 cell c4, c5 cell c5, d cell d, e cell e, f cell f. Bars 50 μm

  • Females fed for 4 days
    • The glandular tissue presents signs of degeneration in all types of acini I, II, and III (Fig. 4f–j). Apoptotic bodies were also found (Fig. 4k).

    • Acini I are intact (Fig. 4e) or in degeneration (Fig. 4f) with reduced lumen.

    • Acini II are in degeneration, present reduced lumen and cells a, c1, c2, and c5 contain secretion (Fig. 4g–h).

    • Acini III are in degeneration and are found with reduced or slightly (Fig. 4i) or very dilated (Fig. 4j) lumen. Those with reduced lumen are more numerous and those that have more dilated lumen are more scarce.

    • Cells d, e, and f are present and f do not contain secretion granules presenting slight signs of degeneration (Fig. 4i–j).

  • Females fed for 6 days
    • The glands present degenerative signs (with numerous cytoplasmic vacuoles, nuclear fragmentation, and presence of apoptotic bodies; Fig. 4m–p) that is more intense than those observed in the females fed for 4 days and subjected to the same glandular extract.

    • Acini I, II, III and indeterminate, all of them are with degenerative characteristics (Fig. 4l–p).

    • Acini I present dilated lumen (Fig. 4l).

    • Acini II present reduced lumen and cells a, c1, c2, c3, and c4 contain secretion granules (Fig. 4m–n).

    • Acini III present reduced lumen and cells d, e, and f (Fig. 4O); f present intense vacuolation in the cytoplasm without secretion granules (Fig. 4o).

    • The results of the glandular alterations found in females fed for 2, 4, and 6 days form groups CG1 and TG are summarized in Tables 1, 2, 3, 4, and 5.

    Table 1

    Comparison of the histological results of the salivary glands of R. sanguineus females from control group (CG1) and test group (TG) with 2 days of feeding

    Acini

    Study groups

    CG1

    TG

     

    SG2

    SGE4

    SGE6

    I

    Intact (Fig. 1a)

    Intact (Fig. 2a–b)

    Intact (Fig. 3a)

    Intact (Fig. 4a)

    Lumen: dilated (Fig. 1a)

    Reduced (Fig. 2a) and dilated (Fig. 2b)

    Dilated (Fig. 3a)

    Reduced and dilated (Fig. 4a)

    II

    Intact (Fig. 1b)

    Intact (Fig. 2b–c)

    Intact (Fig. 3b,d)

    Intact (Fig. 4b,c)

    Lumen: reduced (Fig. 1b)

    Reduced (Fig. 2b,c)

    Reduced (Fig. 3b) and dilated (Fig. 3c–d)

    Reduced (Fig. 4b–c)

    Cells: a, c1, c2, c3, c4, c5 (Fig. 1b)

    Cells: a, c2, c3, c4, c5 (Fig. 2b–c)

    a, c2, c3, c4, c5 (Fig. 3b–d)

    a, c2, c3, c4, c5 (Fig. 4b–c)

    III

    Intact (Fig. 1c)

    Intact (Fig. 2e)

    Intact (Fig. 3e–f)

    Degeneration (Fig. 4i–j)

    Lumen: reduced (Fig. 1c)

    Reduced (Fig. 2e)

    Reduced (Fig. 3e) and dilated (Fig. 3f)

    Reduced (Fig. 4d)

    Cells: d, e, f (Fig. 1c)

    d, e, f (Fig. 2e)

    d, e, f (Fig. 3e–f)

    d, e, f (Fig. 4d)

    Indeterminate

    Absent

    Absent

    Absent

    Absent

    Table 2

    Comparison of the histological results of the salivary glands of R. sanguineus females from control group (CG1) and test group (TG) with 4 days of feeding

    Acini

    Study groups

    CG1

    TG

     

    SG2

    SGE4

    SGE6

    I

    Intact (Fig. 1d–e)

    Intact (Fig. 2f–g)

    Intact (Fig. 3g)

    Intact (Fig. 4e) and in degeneration (Fig. 4f)

    Reduced (Fig. 1d) and dilated lumen (Fig. 1e)

    Reduced (Fig. 2f) and dilated (Fig. 2g)

    Dilated (Fig. 3g)

    Reduced (Fig. 4e–f)

    II

    Intact (Fig. 1e–f)

    Beginning of degeneration (Fig. 2h)

    Degeneration (Fig. 3f–j)

    Degeneration (Fig. 4g–h)

    Lumen: dilated (Fig. 1e–f)

    Reduced (Fig. 2h)

    Reduced (Fig. 3h, j) and dilated (Fig. 3i)

    Reduced (Fig. 4g–h)

    Cells: a, c2, c3, c4, c5 (Fig. 1e–f)

    a, c2, c3, c4 (Fig. 2h)

    a, c1, c2, c3, c5 (Fig. 3h–j)

    a, c1, c2, c5 (Fig. 4g–h)

    III

    Intact (Fig. 1d,g)

    Degeneration (Fig. 2i–j)

    Degeneration (Fig. 3k–l)

    Degeneration (Fig. 4i–j)

    Lumen: reduced (Fig. 1d) and dilated (Fig. 1g)

    Reduced (Fig. 2i) and dilated (Fig. 2j)

    Reduced (Fig. 3k) and dilated (Fig. 3l)

    Reduced (Fig. 4i) and dilated (Fig. 4j)

    Cells: d, e, fa (Fig. 1d,g)

    d, e, fa (Fig. 2i–j)

    d, e, fa (Fig. 3k–l)

    d, e, fa (Fig. 4i–j)

    Indeterminate

    Absent

    Absent

    Absent

    Absent

    aCell without secretion granules

    Table 3

    Comparison of the histological results of the salivary glands of R. sanguineus females from control group (CG1) and test group (TG) with 6 days of feeding

    Acini

    Study groups

    CG1

    TG

     

    SG2

    SGE4

    SGE6

    I

    Intact (Fig. 1h)

    Degeneration (Fig. 2l)

    Degeneration (Fig. 3n)

    Degeneration (Fig. 4l)

    Lumen: dilated (Fig. 1h)

    Dilated (Fig. 2l)

    Dilated (Fig. 3n)

    Dilated (Fig. 4l)

    II

    Intact (Fig. 1i) and in degeneration (Fig. 1j)

    Degeneration (Fig. 2m)

    Degeneration (Fig. 3o)

    Degeneration (Fig. 4m–n)

    Lumen: reduced (Fig. 1i–j)

    Reduced (Fig. 2m)

    Reduced (Fig. 3o)

    Reduced (Fig. 4m–n)

    Cells: a, c1, c2, c3, c4 (Fig. 1i–j)

    a, c2, c3, c4 (Fig. 2m)

    a, c1, c2, c3, c5 (Fig. 3o)

    a, c1, c2, c3, c4 (Fig. 4m–n)

    III

    Degeneration (Fig. 1k–l)

    Degeneration (Fig. 2n–o)

    Degeneration (Fig. 3p)

    Degeneration (Fig. 4o)

    Lumen: reduced (Fig. 1k) and dilated (Fig. 1l)

    Reduced (Fig. 2n) and dilated (Fig. 2o)

    Reduced (Fig. 3p)

    Reduced (Fig. 4o)

    Cells: d, e, fa (Fig. 1k–l)

    e, fa (Fig. 2n–o)

    d, e (Fig. 3p)

    d, e, fa (Fig. 4o)

    Indeterminate

    Scarce (Fig. 1m)

    Scarce (Fig. 2p)

    Numerous (Fig. 3q)

    Numerous (Fig. 4p)

    aCell without secretion granules

    Table 4

    Comparison of the result of eosin staining of secretion granules of cells a and c5 of acini II and d and e of III of the salivary glands of females from CG1 and TG fed for 2, 4, and 6 days

    Acini

    Period of feeding

    Study groups

    CG1

    TG

     

    SGE2

    SGE4

    SGE6

    II

    2 Days

    a (5) (Fig. 1a–b)

    a (5) (Fig. 2b–d)

    a (5) (Fig. 3b–d)

    a (6) (Fig. 4b–c)

    c5 (3) (Fig. 1a–b)

    a (4) (Fig. 2b–d)

    c5 (3) (Fig. 3b–d)

    c5 (4) (Fig. 4c)

    4 Days

    a (3) (Fig. 1e–f)

    a (6) (Fig. 2h)

    a (4) (Fig. 3h–j)

    a (7) (Fig. 4g–h)

    c5 (2) (Fig. 1e)

    c5a

    c5 (2) (Fig. 3j)

    c5 (3) (Fig. 4g)

    6 Days

    a (3) (Fig. 1i–j)

    a (7) (Fig. 2m)

    a (5) (Fig. 3o)

    a (7) (Fig. 4m)

    c5a

    c5a

    c5a

    c5a

    III

    2 Days

    d (5) (Fig. 1c)

    d (5) (Fig. 2e)

    d (5) (Fig. 3e–f)

    d (5) (Fig. 4d)

    e (3) (Fig. 1c)

    e (3) (Fig. 2e)

    e (4) (Fig. 3e–f)

    e (4) (Fig. 4d)

    4 Days

    d (3) (Fig. 1d, g)

    d (5) (Fig. 2i–j)

    d (5) (Fig. 3k–l)

    d (4) (Fig. 4i–j)

    e (3) (Fig. 1d, g)

    e (2) (Fig. 2i–j)

    e (5) (Fig. 3k–l)

    e (3) (Fig. 4i–j)

    6 Days

    d (3) (Fig. 1l)

    da

    d (5) (Fig. 3p)

    d (4) (Fig. 4o)

    e (0) (Fig. 1k–l)

    e (1) (Fig. 2n–o)

    e (6) (Fig. 3p)

    e (4) (Fig. 4o)

    (0) no staining, (7) strongly positive staining

    aCell not observed

    Table 5

    Comparison of the intensity of the degenerative process in the salivary glands of R. sanguineus females from groups CG1 and TG fed for 2, 4, and 6 days

    Salivary glands

    Study groups

    Intensity of glandular degeneration

    2 Days

    CG1

     

    0 (Fig. 1a–c)

    TG

    SGE2

    0 (Fig. 2a–e)

    SGE4

    0 (Fig. 3a–e)

    SGE6

    0 (Fig. 4a–e)

    4 Days

    CG1

     

    0 (Fig. 1d–g)

    TG

    SGE2

    1 (Fig. 2f–l)

    SGE4

    2 (Fig. 3g–m)

    SGE6

    3 (Fig. 4e–k)

    6 Days

    CG1

     

    4 (Fig. 1h–n)

    TG

    SGE2

    5 (Fig. 2l–q)

    SGE4

    6 (Fig. 3n–r)

    SGE6

    7 (Fig. 4l–q)

    (0) absence of degenerative characteristics, (7) more intense degenerative characteristics

Discussion

The present study provided histological results of salivary glands of R. sanguineus females fed for 2, 4, and 6 days in rabbits which were previously immunized with glandular extracts SGE2–SGE6. It is clear that the resistance of the host influenced in the secretory cycle of the infesting females glands, acting both in the secretory activity of the glandular cells (making them inactive) and leading the glands to early degeneration. Nunes et al. (2010) and Jittapalapong et al. (2008) have reported that the resistance acquired by the hosts studied by them induced the salivary glands of Amblyomma cajennense and R. (Boophilus) microplus females, respectively, to necrosis. On the other hand, the present study showed that the resistance of the host led to the loss of function (inactivity) of some secretory cells as well as to the early degeneration of the salivary glands due to the occurrence of apoptosis. However, it is known that different species of ticks respond differently to the resistance acquired by the host (Nunes et al. 2010).

The results here obtained showed that under the histological point of view, the glands of females fed for 2, 4, and 6 days from CG1 (control) are similar to those from CG2, demonstrating that Freund’s adjuvant, used with glandular extracts did not influence the glandular physiology. As for the glandular extracts, it is clear that they would induce the development of resistance by the hosts, which affected the glandular physiology, i.e., the glands had the secretory activity reduced, becoming less efficient when compared to individuals from CG1, data that corroborate Jittapalapong et al. (2008), who reported that the immunization of the host with glandular extracts would affect directly the salivary glands of the infesting ticks, which would make the feeding process less efficient and would possibly reduce the transmission of pathogens.

The results here presented showed that the resistance acquired by the hosts immunized with SGE2, SGE4, and SGE6 extracts affected much more acini II and III cells than those of acini I. This has probably occurred because these cells present secretory function, while those of acini I act on the osmoregulation and in the hydric balance of the ectoparasite (Binnington 1978; Walker et al. 1985). Thus, cells of acini II and III are responsible for the secretion of antigenic molecules and shape the immune-inflammatory system of the host, corroborating Jittapalapong et al. (2008) and Nunes et al. (2010) who also demonstrated that acini II and III cells were more affected by the resistance of the host.

Adding to this, Ferreira et al. (1996) demonstrated that acini II and III of R. sanguineus ticks would be the ones that synthesize more and secrete antigenic molecules which would cause the development of elevated immunologic response of the hosts. Gill et al. (1986) reported the occurrence of intense immunological response to some glandular antigens of Hyalomma anatolicum anatolicum tick, such as glycoproteins, acid phosphatase, esterase and aminopetidase, molecules synthesized mainly by the glandular cells of acini II and III (Binnington 1978; Walker et al. 1985).

Other important point considered in the present study was the resistance of the hosts of each of the GT groups affected the salivary glands of females fed for 2, 4, and 6 days. In those fed on hosts immunized with SGE2 and SGE4, there were alterations in cells of acini II and III, while in those subjected to SGE6 only those of II were affected, demonstrating that the immune system of the hosts responded in a different and specific way to the immunization with different extracts.

The resistance to extract SGE2 acted on the secretory activity of cells c1 of acini II, which were inactive for three glandular periods, c5 of acini II, which were inactive in the glands of females fed for 4 days and in cells d of acini III, which would stay inactive in the glands of females fed for 6 days. As for the resistance to SGE4, it acted on cells c1 and c4 of acini II and f of acini III. Cells c1 were inactive in glands of 2 days and started to present activity in those of 4 days and 6 days. Cells c4 were active in glands of 2 days, lost activity in those of 4 days and became active again in glands of 6 days. As for cells f, they were active in glands of 2 and 4 days and became inactive in those of 6 days.

The resistance acquired with the immunization with extract SGE6 acted only on cells c1, c3, c4, c1 were inactive in the glands of 2 days and started to present activity in those of 4 and 6 days. Cells c3 e c4 were active in glands of 2 days, became active in those of 4 and start to present activity again in those of 6 days.

It was demonstrated here that the glandular cells that had their secretory performance more affected by the resistance of the host were c1. The c4 presented intermediate alterations, followed by c3, c5, d, and f. Data here obtained about cells from group c and f corroborate those obtained by Jittapalapong et al. (2008), who showed that these cells would be very affected by the resistance of the host and the study by Nunes et al. (2010) corroborated data here obtained for cells c1, c3, and c4, once according to the authors the cells from group c would also be very affected by the action of the resistance of the host.

Here, the fact that cells c1, c3, c4, c5, d, and f were affected by the resistance indicated that the secretions produced by them would be highly immunogenic in relation to those from other types of cells present in the salivary glands. In addition, the alterations suffered by cells c1, c3, c4, c5, d, and f caused alterations in the glandular secretory process, reducing it, which caused a reduction in the feeding efficiency of R. sanguineus females and demonstrated that such cells would possibly play an important role in the modulation of the host. Differently from what was here demonstrated, Almeida et al. (1994) reported that the molecules from cells a, d, and e would be the most immunogenic, while those from group c would not present this characteristic.

The fact that the secretions of cells c1, c3, and c4 are related to the modulation of the host corroborates Binnington (1978) and Walker et al. (1985), who reported that the cells from group c would act in the manipulation of the response of the host. According to Binnington (1978), cells c1 of acini II of the salivary glands of Boophilus microplus females would be responsible for the production of esterase, which, according to Geczy et al. (1971) would act in the increase of the vascular permeability and in the hydrolisis of cholesteryl ester.

From the analysis of the glands of females fed for 4 and 6 days on host immunized with extract SGE4 important information was obtained about cells c4 concerning their participation in the modulation of the host. It was observed that these cells were inactive in glands of 4 days, impairing their glandular performance, which made the ectoparasite’s feeding deficient. This can be proved by the fact that cells f of glands of 6 days were inactive, because in normal conditions they would be hypertrophied due to their intense osmoregulating action, which would occur due to the full consumption of blood by the tick, making it clear that the secretion produced by cells c4 would present, in addition to others, the function of modulating the vasodilating mechanisms and coagulation of the host’s blood, which would allow the great consumption of blood by the ectoparasite.

Although literature has indicated that d cells are involved with the fabrication of components of the cement cone during the attachment of the tick (Walker et al. 1985; Gill and Walker 1987), the results here obtained demonstrated that, in R. sanguineus females, the secretion produced by these cells also play a role in the modulation of the host, once they were affected by the resistance acquired by the animals immunized with SGE2 extract. Binnington (1978), studying the salivary glands of B. microplus females, observed that cells d would also synthesize and secrete acid phosphatase, enzymes that catalyze the molecules break (digestion). Thus, considering what has previously been reported, it can be inferred that the secretion released by cells d of R. sanguineus females would also be acting in the digestion of the tissues of the host’s skin in order to maintain the feeding lesion open.

In addition to the ability of resistance of the hosts from group TG in making some glandular cells inactive, it is clear that this resistance affected the glandular cells a and c5 of acini II and d and e of acini III in a way that they altered the composition of the synthesized secretion, alteration reflected in the histology and that varied both according to the study group and to the period of feeding of the females, allowing the assumption that the secretion synthesized by such cells would be immunogenic and would probably act in the modulation of the host. According to Binnington (1978), Walker et al. (1985), Fawcett et al. (1986), and Gill and Walker (1987), the secretions produced by cells a, d, and e (lipoprotein nature) would participate in the formation of the cement cone; however, it is known that the present other molecules in their composition such as acid phosphatase, catechol oxidase (cells a), and aminopeptidase (cells d and e; Binnington 1978; Walker et al. 1985). Walker et al. (1985) discussed the possibility of aminopeptidase acting on the facilitation of the infiltration of the cement in the feeding lesion, acting directly in the organism of the host. Thus, the fact that the secretions produced by cells d and e play a role in the immunogenicity can be explained.

In the present study, it was also verified that the resistance of the host, induced by glandular extracts SGE2–SGE6, led the glands of females fed for 4 days to degeneration, which would be precocious in relation to group CG1, where this alteration only took place in the glandular tissue of 6-day fed females. These data corroborate those by Jittapalapong et al. (2008) who demonstrated that the immunization of the hosts with glandular extracts would precociously induce the degeneration of the salivary glands of R. (Boophilus) microplus females. In addition, Nunes et al. (2010) also reported that the resistance acquired by successive infestations would also induce salivary glands of A. cajennense females to early degeneration.

A possible explanation for the early induction of degeneration would be the mechanism which controls the synthesis of ecdisteroids, hormone which stimulates the glandular degeneration (Mao et al. 1995; Lomas et al. 1998; Weiss and Kaufman 2001), which was intensively synthesized in females from group TG (SGE2 and GS24), fed for 4 days, a precocious synthesis in relation to what was obtained in those exposed to normal conditions (occurring in the end of the feeding process; Lomas 1993).

The information above is probably explained by the acquisition of resistance by the immunized hosts, which reduced the infesting ectoparasites’ blood consumption by making it more difficult (Furquim et al., submitted paper). Even with the reduction of blood ingestion these females would reach the “critical weight” (increase in ten times the weight of a fasting tick) which would trigger the initial production of ecdysteroid, as would happen in normal conditions (Weiss and Kaufman 2001). However, upon reaching 4 days of feeding they would not be able to reach the stage of rapid engorgement (consumption of a large volume of blood in a short period of time), which was proved by the need of a longer time to reach complete engorgement (Furquim et al., submitted paper).

Because of the fact that the females had not started the phase of rapid engorgement, the rest of the feeding process was impaired, and upon arriving on the sixth day of feeding did not present intense consumption of blood (Furquim et al., submitted paper) demonstrated by: (a) the weight of the completely engorged females subjected to extracts SGE2 and SGE4 was inferior to those from group CG (Furquim et al., submitted paper) and (b) the histology of the glandular cells of these females showed acini III with less dilated lumen in relation to those from CG1, a result of the less intense actuation in the osmoregulating process of the ticks (Fawcett et al. 1986; Sonenshine 1991; Sauer et al. 2000).

The fact that the females did not present intense consumption of blood showed that their feeding process was not sufficiently able to fulfill all the needs, such as provide the necessary conditions to make the vitellogenic process successful (Rosell-Davies and Coons 1989), fact that was proven by data obtained by the reduction of the eggs mass as well as by the minor feasibility of the eggs in comparison to those from CG1 (Furquim et al., submitted paper). Thus, the nervous system of the females would be activated and the synthesis of ecdysteroid already initiated, would be intensified, increasing the concentration of this hormone in the hemolymph and stimulating the glandular degeneration (Lomas 1993; Weiss and Kaufman 2001).

Possibly, the physiological mechanism which led the salivary glands of R. sanguineus females subjected to SGE2 and SGE4 extracts to degenerate earlier in comparison to those from CG1 is similar to the one of Amblyomma hebraeum females, where the females were removed from the host after having reached “critical weight”, meaning that they did not present intense blood consumption, which resulted in a power body weight, continuous increase in the synthesis of ecdysteroid, leading the salivary glands to early degeneration in addition to reducing oviposition (Weiss and Kaufman 2001).

On the other hand, the stimulus for the glandular degeneration of females fed on hosts immunized with SGE6 were more significant, i.e., it was even earlier than in females subjected to extracts SGE2 and SGE4, suggesting that the occurrence of a mechanism completely opposite to the one described above. The fact that these females presented high consumption of blood and consequent increase in weight in a short period of time (Furquim et al., submitted paper), stimulated the early production of ecdysteroids, which made the process of glandular degeneration also precocious.

Thus, according to the results obtained here, the immune-inflammatory responses developed by the hosts immunized by the different glandular extracts made the occurrence of the glandular degeneration process precocious in relation to the control group (CG1). However, such responses of the hosts do not alter other characteristics observed in R. sanguineus females in normal conditions (Furquim et al. 2008). In this sense, it was verified that in the glandular tissue of the females from TG that the degenerative assynchronism was maintained, where acini II and III were the most affected in relation to acini I, data which corroborate Nunes et al. (2010). According to these authors, the glandular degeneration induced by the resistance of the host would maintain the same pattern.

In addition, in the salivary gland of females from TG the most affected cells of acini II and III were those from groups c and f, which presented intense cytoplasmic vacuolation, corroborating Jittapalapong et al. (2008), who reported that cells with more intense degenerative characteristics were c (agranular) and f.

Other degenerative glandular characteristics maintained in the glands of GT in comparison to CG1 were: loss of cellular contact, cytoplasmic vacuolation, nuclear changes (picnosis and fragmentation), as well as formation of apoptotic bodies characterizing the occurrence of atypical apoptosis, according to what was previously suggested by Furquim et al. (2008) for completely engorged R. sanguineus females 3 days after engorgement in normal conditions. Although Jittapalapong et al. (2008) and Nunes et al. (2010) have observed in their studies the occurrence of degenerative characteristics in the glandular cells similar to those here verified, they suggested that the acquisition of resistance by the hosts, either by immunization by glandular extracts (Jittapalapong et al. 2008) or successive infestations (Nunes et al. 2010), led the glandular cells of R. (Boophilus) microplus and A. cajennense females to necrosis.

Thus, it was here established that the immunization of hosts with SGE2 and SGE4 glandular extracts of R. sanguineus females have influenced negatively in the secretory cycle of salivary glands, reducing their secretory ability and consequently reducing the ectoparasites feeding (making it less efficient) important information for the better understanding and creation of alternatives to control these ectoparasites.

Acknowledgments

This research has been supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo; grants no. 08/58443-7 and 07/59020-0) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; grant no. 308733/2006-1 and M.I. Camargo-Mathias and G.H. Bechara academic carrier research fellowships). Part of this work has been facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3) supported by the European Union under contract number 510561-INCO.

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