Parasitol Res (2004) 94: 304–311 DOI 10.1007/s00436-004-1215-4
O R I GI N A L P A P E R
Irina S. Khokhlova Æ Marina Spinu Æ Boris R. Krasnov A. Allan Degen
Immune responses to fleas in two rodent species differing in natural prevalence of infestation and diversity of flea assemblages Received: 21 June 2004 / Accepted: 11 August 2004 / Published online: 15 September 2004 Springer-Verlag 2004
Abstract We studied in vitro immune response to fleas in two gerbils, Gerbillus dasyurus and Gerbillus andersoni allenbyi, which differed in their natural species richness of flea assemblages and prevalence of infestation. G. dasyurus is parasitized naturally by several flea species, but the prevalence of infestation is low, whereas G. a. allenbyi is parasitized by a single flea species, with high prevalence of infestation. We hypothesized that immunological parameters and the cell-mediated specific immune response to an antigen from an unfamiliar flea species differ between the two gerbil species. Parasitized and control gerbils of both species demonstrated similar, relatively low levels of spontaneous glucose consumption. The same was true for the phytohemagglutinin treatment. Responses to antigen from unfamiliar flea species were higher than both spontaneous glucose consumption and response to phytohemagglutinin in parasitized and control G. a. allenbyi and parasitized G. dasyurus. However, no significant difference in the spontaneous blast transformation index and responses to both phytohemagglutinin and flea antigen was found in control G. dasyurus. The number of white blood cells was significantly lower in control than in parasitized G. dasyurus, whereas no difference in the number of white blood cells was found between control and parasitized G. a. allenbyi. The levels
I. S. Khokhlova Æ A. A. Degen Wyler Department of Dryland Agriculture, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, Israel M. Spinu University of Agricultural Sciences and Veterinary Medicine, Manastur str. 3–5, Cluj-Napoca, Romania B. R. Krasnov (&) Ramon Science Center and Mitrani Department of Desert Ecology, Jacob Blaustein Institute for Desert Research, Ben-Gurion University of the Negev, P.O. Box 194, 80600 Mizpe Ramon, Israel E-mail:
[email protected] Tel.: +972-8-6586337 Fax: +972-8-6586369
of circulating immune complexes and concentrations of immunoglobulins did not differ between parasitized and control individuals in both species. Phagocytic activity was significantly higher in males than in females of G. a. allenbyi but not of G. dasyurus. In addition, phagocytes of G. dasyurus appeared to be significantly more active than those of G. a. allenbyi.
Introduction A parasite is commonly defined as ‘‘an organism that lives in or on a host from which it derives its food and other biological supplies’’ (Kim 1985). The host, therefore, represents a habitat for a parasite, providing it with food resources and space to live, feed and mate. By exploiting a host, parasites exert negative effects on it by depleting its energy and nutrients. In contrast to habitats of free-living organisms, habitats of parasites (=hosts) are not submissive victims of their parasites, but defend themselves actively using specific behavioral (e.g., autogrooming; Mooring et al. 2000), physiological (e.g., elevated body temperature; Banet 1986) and/or immunological mechanisms. The immune system is the primary means by which the host can protect itself from a parasite. This system is aimed to discriminate between ‘‘self’’ and ‘‘non-self’’, and to minimize the consequences of contact with foreign molecules introduced into the host by the feeding parasites. Activation of an immune response, and even maintenance of a competent immune system are an energetically demanding, protective process that requires tradeoff decisions among competing energy demands for growth, reproduction, thermoregulation, work, and immunity (Sheldon and Verhulst 1996, but see Klaising 1998). In other words, the tradeoffs occur between defense against parasites and other concurrent needs of the organism (Sheldon and Verhulst 1996). Empirical evidence suggests that such costs can be relatively high (e.g., Moret and Schmid-Hempel 2000). As a result,
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many hosts generally have low circulating titers of immune effectors such as leukocytes, immunoglobulins and complement (Klein 1990). Development of immune responses and investment into immune defenses should depend on the pattern of parasite pressure (see Combes 2001 and references therein). For example, it can depend on the frequency and probability of parasite attacks (Martin et al. 2001; Tella et al. 2002). Selection of mechanisms of resistance in hosts is expensive, and thus of little advantage if encounters with the parasite are rare (Poulin et al. 1994). Consequently, if frequency and/or probability of attacks by parasites are low, then a host can limit its allocation of energy for immune responses by the development of the responses only after being attacked by a parasite (‘‘post-invasive’’). If, however, frequency and/ or probability of parasitism are high, an advantageous strategy of a host can be high investment into immune defense and, thus, continuous maintenance of a certain level of immune ‘‘readiness’’ (Jokela et al. 2000). In addition, the long and continuous association between a particular host and a particular parasite can induce host genotypic changes via selection. In particular, these changes could affect the major histocompatibility complex that is the region of the genome that controls immune responses (Gruen and Weissman 1997). As a result of any of these processes, even those host individuals that have never been attacked can, nevertheless, maintain some protection against a parasite whose attack is highly probable (Jokela et al. 2000). Another component of parasitism that can affect the pattern of development and persistence of defensive responses in hosts is a degree of variety of parasite challenges. Maintaining several different means of defense is likely more costly than sustenance of one specific type of defense (Taylor et al. 1998). Consequently, host species that are exploited by a small number of specific parasites can acquire specific immune resistance against these parasites but not against other, albeit phylogenetically related parasites. In contrast, hosts with a diverse parasite spectrum can develop multiple immune responses against a variety of parasite species. As a result, mounting of the immune responses to non-familiar parasites should be expected in a ‘‘parasite-rich’’ rather than in a ‘‘parasite-poor’’ host. Fleas (Siphonaptera) are obligate ectoparasites of higher vertebrates, being most abundant and diverse on small to medium-sized burrowing mammalian species. Fleas have a negative effect on life history traits and reproductive success of their hosts (Lehmann 1993; Richner et al. 1993). For example, flea parasitism was shown to affect energy requirements of the desert gerbil Gerbillus dasyurus (Khokhlova et al. 2002). Moreover, the major effects of these parasites on the energy expenditure of this host seemed to be through factors other than blood deficiency, such as energy allocation to immune reaction or to easing the irritative effect (Khokhlova et al. 2002). In this study, we examined in vitro immune responses to fleas in two closely related desert gerbils, Gerbillus
dasyurus and Gerbillus andersoni allenbyi, which differ in their natural species richness of flea assemblages and prevalence of infestation. G. dasyurus and G. a. allenbyi are common rodent species of the Negev desert, Israel. G. dasyurus (adult body mass 23–29 g) occupies a variety of habitats and is parasitized naturally by several flea species (Xenopsylla dipodilli, Xenopsylla conformis, Xenopsylla ramesis, Nosopsyllus iranus theodori, Stenoponia tripectinata, Coptopsylla africana, Rhadinopsylla masculana), although species composition of flea assemblages on this species varies among habitats (Krasnov et al. 1997). In particular, X. conformis and X. ramesis replace each other on G. dasyurus between two different habitats situated at opposite ends of a steep precipitation gradient (Krasnov et al. 1998). Prevalence of infestation of G. dasyurus by fleas varies among habitats in the range 20–65%, and never attains higher values (Krasnov et al. 1998). Intensity of G. dasyurus infestation by fleas also differs among habitats, ranging from a low of 2.0 fleas per infested individual to a high of 6.3 fleas per infested individual (Krasnov et al. 1998). In contrast, G. a. allenbyi (adult body mass 25–30 g) is a specialist sanddweller and is parasitized mainly by a single flea species, Synosternus cleopatrae pyramidis (Krasnov et al. 1999). Prevalence of G. a. allenbyi infestation by fleas is 95–100%, whereas intensity of infestation averages 12.2 fleas per infested individual (H. Tzairi, Z. Abramsky and B. Krasnov, unpublished data). We hypothesized that immunological parameters as well as the pattern of immune responses to an antigen from an unfamiliar flea species differ between G. dasyurus and G. a. allenbyi. We predicted that non-parasitized G. dasyurus will demonstrate lower immune reactions than parasitized conspecifics, whereas the difference in the manifestation of these reactions between non-parasitized and parasitized G. a. allenbyi will be less pronounced. We also predicted that immune responses to antigen from an unfamiliar flea will be less pronounced in G. a. allenbyi than in G. dasyurus.
Materials and methods Rodents We used rodents from our laboratory colonies. Progenitors of the G. dasyurus colony were captured at the Ramon erosion cirque, Negev Highlands, Israel (3035¢N, 3445¢E) in 1996. We used individuals whose progenitors originated from areas where G. dasyurus is infested with X. dipodilli, X. c. mycerini, N. i. theodori, and C. africana, but never infested with X. ramesis. Progenitors of the G. a. allenbyi colony were collected at two locations in the western Negev, Beer Malaga (3056¢N, 3424¢W) and Retamim, Israel (3104¢N, 3441¢E) in 2000. Animals were maintained either in outdoor enclosures (3·2·2 m) or in animal rooms. The enclosures were built of wire mesh (1·1 cm), and contained a 60-cm layer of either natural sandy-loess–
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gravel substrate (for G. dasyurus) or natural sand (for G. a. allenbyi), which allowed rodents to burrow. Millet seed and alfalfa (Medicago sp.) leaves were provided daily ad libitum. The enclosure populations were started with 10 or 20 individuals (G. dasyurus, five males and five females, or G. a. allenbyi, eight males and 12 females), all of which were infested with ten X. dipodilli each (G. dasyurus) or with ten S. c. pyramidis each (G. a. allenbyi). At the time of the experiments, there were 40 G. dasyurus and 30 G. a. allenbyi in the enclosures. Prevalence and intensity of flea infestation in the enclosures at that time were 57.1% and one to two fleas per infested individual for G. dasyurus, and 100% and two to three fleas per infested individual for G. a. allenbyi. Rodents in the animal rooms were housed individually in plastic cages (60·50·40 cm) at 25C, with a photoperiod of 12:12 (L:D) h. They were fed millet seeds and alfalfa ad libitum. Dried grass was provided as bedding material. These animals were not subjected to flea parasitism. Measurements were done when gerbils were 7–19 months old. Immunological studies were done (1) on 35 G. dasyurus (13 males and 23 females) from the enclosure (hereafter referred to as parasitized animals), and on 11 G. dasyurus (six males and five females) maintained in the animal room (hereafter referred to as control animals), and (2) on 23 G. a. allenbyi (14 males and nine females) from the enclosure and on 16 G. a. allenbyi (ten males and six females) from the animal room.
collected and then frozen at )20C. Weighed frozen fleas (100 individuals) were thoroughly stirred in a mortar, mixed with phosphate buffer saline (PBS) at 1.5 times the weight of the fleas, and filtered through plane filter paper to remove remnants of chitin. Then, the extracts were centrifuged for 20 min at 6,000 rpm (CN-2060 microprocessor control centrifuge, Hsiangtai Machinery Industry Co. Ltd., Taiwan), and the pellets of antigen were resuspended in PBS and freeze-dried. Before use, the antigen was diluted with PBS to about 50% of the initial mass of the processed fleas, and sterilized by filtering through 0.2-lm filters (Schleicher & Schuell Inc., Dassel, Germany).
Fleas
White blood cell count
Xenopsylla ramesis is a common ectoparasite of gerbils and jirds throughout the Middle East. We recorded X. ramesis mainly on Meriones crassus, G. dasyurus, Psammomys obesus, and Eliomys melanurus (Krasnov et al. 1997). Fleas for antigen preparation (see below) were obtained from our laboratory colonies, started in 1998– 2001 from field-collected specimens on M. crassus using rearing procedures described elsewhere (Krasnov et al. 2001). In brief, an individual rodent host was placed in a cage that contained a steel nest box with a screen floor and a pan containing a mixture of sand and dried bovine blood (nutrient medium for larvae). Every 2 weeks, all substrate and bedding material were collected from the nest box and transferred into an incubator, where flea development and emergence took place at 25C and 75% relative humidity (RH). The newly emerged fleas were placed on clean animals. Colonies of fleas were maintained at 25C and 75% RH, with a photoperiod of 12:12 (L:D) h.
Two microliters of the heparinized blood were diluted 1:10 with Tu¨rk solution, kept at room temperature for 3 min, and then leukocytes were counted in a Bu¨rkerTu¨rk chamber, in the four corner squares of the grid. The mean value was multiplied by 10 for the dilution degree, and by 10 for the height of the diluted blood layer in the chamber. The values were expressed in number of cells/mm3.
Procedures Whole-body extracts of fleas We prepared whole-body extracts from newly emerged fleas that did not feed after emergence. Fleas were
Blood samples Heparinized (500 IU/ml) blood samples (150 ll) were collected using sterile Pasteur pipettes, from the infraorbital sinus of each rodent. We did not anesthetize the animals because of the negative effect of anesthesia on both the blood (inducing haemolysis) and the recovery duration of the animals (I. S. Khokhlova and M. Spinu, unpublished data). Blood from each animal was sampled once or twice with a 10-day break between samplings. From our observations, rodents recovered fully 1–2 min after blood sampling.
Tests
Leukocyte blast transformation test Mononuclear cells, sensitized in vivo by various antigens, possess the capacity to respond vigorously to the same antigen when contacted in vitro (blast transformation test). In the present study, 120 ll of each heparinized (final concentration of 50 IU/ml) blood sample was mixed with 480 ll of a cell culture medium (RPMI 1640), distributed in equal aliquots in three wells of a sterile, 96-well plate, for the in vitro experimental variants as follows: (1) untreated control culture, (2) phytohemagglutinin-M (PHA; 1 ll per well), and (3) antigen of X. ramesis (2.5 ll per well)-treated cultures. The plates were incubated at 37.5C in a 5% CO2 atmosphere for 15 h. Preliminary studies were conducted to standardize the technique and reagent amounts for the rodent cells. At the end of the incubation period, cell growth was quantified by means of
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a glucose consumption technique. Glucose concentrations were measured in the initial medium and in all the supernatants using a standard (100 mg/dl) glucose solution and a colorimetric test. After mixing 12.5 ll of the culture supernatant with 0.5 ml ortho-toluidine, the samples were boiled for 8 min, cooled quickly in cold running water, and read spectrophotometrically at a wavelength of 610 nm (Unico 2100, United Products Instruments Inc., Dayton, NJ, USA) in 96-well plates (d=1), using the reagent as a blank. The transformation index (TI, %) was calculated as follows: TI= [(MG)SG)/ MG]·100, where MG is the glucose concentration in the initial culture medium, and SG the glucose concentration in the sample after incubation.
at 6,000 rpm for 40 min (CN-2060 microprocessor control centrifuge, Hsiangtai Machinery Industry Co. Ltd., Taiwan), were added to 50 ll heparinized blood. Fifteen microliters of the mixture were transferred immediately to 2 ml of saline, and the rest was incubated for 15 min at 37C. The phagocytic sample removal was repeated after 15 and 30 min. The final tubes, with the mixtures of blood, India ink and saline, were centrifuged at 800 rpm, and the supernatants were read spectrophotometrically (k=535 nm, d=1 cm). There was a decrease in absorbance with time as carbon was phagocytized. The phagocytic activity index was calculated as the difference between the natural logarithms of the optical densities of phagocytosis at 0–15 min and 15–30 min, divided by time (15 min).
Circulating immune complex (CIC) measurements The measurement of circulating immune complexes allows an evaluation of the level of molecular clearance capacity of the body, at a particular moment. Blood samples collected from rodents were allowed to clot (30 min at 37C), and sera were separated by centrifugation (3,000 rpm, 10 min). The samples were kept at )20C until tested. A 4.2% polyethylene glycol (PEG) 6000 solution in borate buffer was used as the precipitating agent, while buffer-treated samples served as controls for borate-induced precipitation. For each sample, 3.3-ll aliquots of the serum were mixed with 196.7 ll borate buffer or PEG solution in parallel wells. The immune complexes precipitate in 60 min at 22–23C. Spectrophotometrical quantification of the precipitates was done at a wavelength of 450 nm in the test plate (d=0.5 cm; multichannel spectrophotometer SUMAL PE2, Karl Zeiss, Jena). CIC concentrations, expressed in optical density units (ODU), were calculated by subtracting the value of the control serum+buffer) from that of the PEG precipitate. Immunoglobulin measurements Total immunoglobulins, known as opsonins, play an important role in innate immunity. Concentrations as low as 24 mg/l of heavy metal salts precipitate the immunoglobulin, affecting their colloidal stability and electric charge. Volumes of 6.6 ll of the sera were diluted in 193.4 ll of a 0.024% barbital-buffer zinc sulfate solution, and allowed to precipitate for 30 min at room temperature (22–23C). The levels of total immunoglobulins were quantified in optical density units (ODU) after spectrophotometrical readings (k=475 nm, d=0.5 cm; multichannel spectrophotometer SUMAL PE2, Karl Zeiss, Jena). Carbon particle inclusion test (phagocytic activity) Phagocytic cells engulf inert particles such as carbon due to the defensive capacity of these cells. Two microliters of supernatant of India ink, obtained by centrifugation
Data analysis Dependent variables did not deviate significantly from normality either before (phagocytic activity, blast transformation index of leucocytes) or after logarithmic (CIC, total immunoglobulin) transformation (ShapiroWilk’s tests, W=0.96–0.98, P>0.3) and, therefore, parametric statistics were applied. Because the same parameter (transformation index) in the presence of two mitogens (PHA and flea antigens) as well as the spontaneous transformation index were measured in each individual, we analyzed the effects of species, sex and parasitological experience (previously parasitized versus control) of a rodent on immune responses using repeated-measures ANOVA with the transformation index being a within-subjects factor, and species, sex and parasitological experience being between-groups factors. The effects of sex and parasitological experience on nonspecific immune responses were analyzed for each species separately, using two-way ANOVAs with immunological parameters as dependent variables, as well as three-way ANOVAs for both species together. Tukey’s honest significant difference (HSD) tests for unequal samples were applied for all multiple comparisons. To avoid an inflated type I error, we applied Bonferroni adjustment of alpha (significance was accepted at the adjusted alpha level of 0.0125). Data are presented as means±SE.
Results Transformation indices of leucocytes differed significantly in different treatments (univariate test of significance for planned comparison, F=2,489.2, P0.1 for both; Table 1). Furthermore, the pattern of responses of control and
308 Table 1 Summary of the repeated measures ANOVA of the results of the leucocyte blast transformation test in dependence on species, sex, and parasitological experience of rodents and treatment (control, PHA, flea antigen)
Effect
Sum of squares
df
F
P
Species Sex Parasitological experience Species·sex Species·parasitological experience Sex·parasitological experience Species·sex·parasitological experience Error Treatment Treatment·species Treatment·sex Treatment·parasitological experience Treatment·species·sex Treatment·species·parasitological experience Treatment·sex·parasitological experience Treatment·species·sex·parasitological experience Error
0.02 0.0003 0.005 0.01 0.06 0.02 0.01 2.41 0.30 0.0005 0.002 0.01 0.005 0.013 0.002 0.003 0.21
1 1 1 1 1 1 1 76 2 2 2 2 2 2 2 2 144
0.63 0.01 0.13 0.27 1.61 0.62 0.35
0.43 0.92 0.72 0.60 0.21 0.43 0.55
101.3 0.17 0.66 4.59 1.79 4.29 0.53 0.85
>0.001 0.84 0.52 0.01 0.17 0.01 0.59 0.43
parasitized animals differed between species. This explains the significance of the interaction terms of treatment·parasitological experience, and treatment· species·parasitological experience. Males and females of both species demonstrated similar, relatively low levels of spontaneous glucose consumption, independent of their parasitological experience (Tukey’s HSD tests, P>0.7). The same was true for the transformation index under phytohemagglutinin treatment (Tukey’s HSD tests, P>0.8). Responses to antigen from X. ramesis were higher than both spontaneous glucose consumption and response to phytohemagglutinin in parasitized and control G. a. allenbyi and parasitized G. dasyurus (Tukey’s HSD tests, P0.5 for both; Fig. 2). In addition, parasitized G. dasyurus and all G. a. allenbyi did not differ in this parameter (Tukey’s HSD tests, P>0.3). The level of circulating immune complexes did not differ between parasitized and control individuals in both species rodents (F1,41=0.01 for G. dasyurus, and F1,35=0.91 for G. a. allenbyi, P>0.4 for both). The same was true for between-sex (F1,41=1.89 for G. dasyurus, and F1,35=0.15 for G. a. allenbyi, P>0.2 for both) and between-species (F1,77=2.51, P>0.1) differences. The concentration of immunoglobulins was similar in the
Fig. 1 Mean (±SE) transformation index of leukocytes of G. dasyurus and G. a. allenbyi. SGC Spontaneous glucose consumption, PHA glucose consumption under phytohemagglutinin treatment, FA glucose consumption under treatment with antigen from a flea X. ramesis
Fig. 2 Mean (±SE) number of white blood cells (per mm3) in parasitized and control G. dasyurus and G. a. allenbyi
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parasitized and control G. dasyurus and G. allenbyi (F1,41=0.68 and F1,35=0.29, respectively, P>0.4 for both), as well as in males and females of both species (F1,41=1.91 and F1,35=2.42, respectively, P>0.1 for both) and between species (F1,77=1.99, P>0.1). No effect of parasitological experience on phagocytic activity was found in either G. dasyurus (F1,41=2.76, P>0.1) or G. a. allenbyi (F1,35=0.001, P>0.9). Betweensex difference in phagocytic activity was found in G. a. allenbyi (F1,35=6.4, P0.1). This parameter was significantly higher in males than in females of the former species (Fig. 3). In addition, phagocytes of G. dasyurus appeared to be significantly more active than those of G. a. allenbyi (F1,77=33.81, P
