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Behavioural Brain Research 166 (2006) 236240

Research report

Lateralised behaviour and immune response in dogs: Relations between paw preference and interferon-, interleukin-10 and IgG antibodies production
A. Quaranta a, , M. Siniscalchi a , A. Frate a , R. Iacoviello a , C. Buonavoglia b , G. Vallortigara c
b a Department of Animal Production, University of Bari, Strada Prov.le per Casamassima, Km 3-70010 Valenzano (BA), Italy Department of Animal Health and Well-being, University of Bari, Strada Prov.le per Casamassima, Km 3-70010 Valenzano (BA), Italy c Department of Psychology and B.R.A.I.N. Centre for Neuroscience, University of Trieste, Via S. Anastasio, 12-34123 Trieste, Italy

Received 16 June 2005; received in revised form 6 August 2005; accepted 8 August 2005 Available online 12 September 2005

Abstract The production of specic antibodies (IgG), interleukin-10 (IL-10) and interferon- (IFN-) was evaluated in dogs in relation to behavioural lateralisation as assessed by paw preference. Left-handed, right-handed and ambidextrous dogs of mixed breed were selected on the basis of their performance in a task consisting of the removal of a piece of adhesive paper from the snout. All dogs were immunised with rabies vaccine. IgG anti-rabies antibody response was evaluated by indirect immunouorescence (IIF) test. Serum IFN- and IL-10 levels were measured by ELISA in animals showing signicant individual left-, right- or no-paw preferences in the behavioural test. The results showed that the direction of behavioural lateralisation inuenced the immune response in dogs. The titers of anti-rabies antibodies were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. Similarly, the IFN- serum levels were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. IL-10, on the contrary, seemed to be an immune parameter, which was not affected by lateralisation. These ndings suggest that immunomodulation can be correlated with brain laterality in canine species by the regulation of the production of antibodies and some cytokines like IFN-, which are molecules involved in the immuneneurohumoral crosstalk. 2005 Elsevier B.V. All rights reserved.
Keywords: Lateralisation; Immunity; Dog; Pawedness; Behaviour

1. Introduction Considerable experimental evidence indicates that behavioural and brain lateralisation is widespread in the animal world, suggesting that cerebral functional asymmetry may be a fundamental feature of all vertebrate brains [9,10,1822]. Lateral biases in behaviour (e.g., spontaneous rotational behaviour, paw preference) have been related to immune asymmetries in different animal models, suggesting that the brain modulates the immune system in an asymmetrical way [4,7,8,11]. Using spontaneous rotational behaviour

Corresponding author. Tel.: +39 040 4679927; fax: +39 040 4679883. E-mail address: a.quaranta@veterinaria.uniba.it (A. Quaranta).

in a circular cage as an index of lateralisation, it has been shown that differential reactivity in humoral and cellmediated immune responses of male mice is associated with behavioural sidedness [11]. Mice with left-turning preference had lower in vivo primary IgM and IgG anti-keyhole limpet hemocyanin (KLH) antibody response, delayed-type hypersensitivity response, and host-resistance against the intracellular bacteria Listeria monocytogenes, than mice with right-turning preference. The weak innate immune response of left-turners for clearance of L. monocytogenes showed close interaction with elevated serum IL-6 levels [11]. In mice it has been conrmed that there is also an association between paw preference in food-reaching task and some immune parameters, including natural killer cell activ-

0166-4328/$ see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2005.08.001

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ity, cytotoxic T lymphocyte activity, mixed lymphocyte reaction, mitogen-induced lymphoproliferation [1,7,14,15] and auto-antibody and interleukines production [5,8]. Increased plasma levels of IL-1beta after an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) were observed in left-pawed and ambidextrous mice, but not in right-pawed mice. As to IL-6, the LPS-induced increase was higher in ambidextrous than in left- and right-pawed animals [8]. In dogs, a link between cerebral lateralisation and immune system was rst demonstrated using paw preference in a task consisting of the removal of a piece of adhesive paper from the snout [17]. Population lateralisation was observed, but in opposite directions in the two sexes: males preferentially used their left paw, females their right paw. Nevertheless, no association was detected between sex and paw preference with respect to immune response. In both sexes, left-pawed dogs showed higher percentage of lymphocytes than right and ambidextrous dogs, whereas granulocytes percentage was lower in left-pawed than in right-pawed and ambidextrous dogs. Moreover, the total number of lymphocytes cells was higher in dogs that preferentially used their left paw than in dogs that preferentially used their right paw and ambidextrous dogs, whereas the number of gamma-globulins was lower in left-pawed than in right-pawed and ambidextrous dogs [17]. The aim of this paper is to investigate laterality effects on the production of specic antibodies, interleukin-10 (IL-10) and interferon- (IFN-), which represent one of the factors responsible of brainimmune interactions, induced by rabies vaccine in dogs selected for their paw preference.

Fig. 1. Position of the adhesive tape.

2.2. Paw preference test The test consisted of the removal of an adhesive tape (3MTM Scotch MagicTM 810) of three different sizes (19 mm 38 mm; 10 mm 20 mm; 5 mm 10 mm, adjusted for the size of different animals) located longitudinally to the midline on the nasal bridge in such a way that it could not be seen by the animal [17] (see Fig. 1). The test was carried out in an open eld within the Clinic, which was always the same for all the subjects. Animals were given a total of 10 trials at monthly intervals. The trial consisted of the application of the adhesive tape (by two experimenters, one right-handed and one left-handed who alternated in the task) and then the following behaviours were recorded: (1) the rst paw used in attempts to remove the tape and (2) the total number of attempts made with the left and the right paw in the 2 min of test duration. The behaviour of the animals was recorded continuously on video during the 2 min of test. Individual lateralisation was calculated in two ways, as follows:

2. Materials and methods 2.1. Animals Thirty-six dogs (18 intact males, 18 intact non-oestrus females) of mixed breed, aged between 1 and 5 years, were tested for their paw preference (see below). All of dogs were family pets whose owners had consented to participate in the experiment during periodic visits to the Faculty of Veterinary Medicine of Bari, Italy. No subject was previously trained. Coprological analysis for parasites in addition to clinical examination was carried

Number of times the left paw was used rst 100 Number of times the left paw was used rst + Number of times the right paw was used rst Total number of times the left paw was used during the test 100 Total number of times the left paw was used during the test + Total number of times the right paw was used during the test out for all animals. Moreover, all animals were vaccinated against the most common diseases (Vanguard 7 , Pzer, Italy) 68 months earlier. No dogs were previously vaccinated against rabies.

(1)

(2)

Animals were selected on the basis of a signicant individual preference (estimated by one-tailed binomial test, P < 0.05) for using a particular paw for the initial attempt to remove the adhesive paper and for using a particular paw in the total number of attempts during the 2 min of test.

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Fig. 2. Anti-rabies antibody production in right-, left- and ambi-preferent dogs (group means with S.E.M. are shown).

2.3. Anti-rabies antibody evaluation by the indirect immunouorescence (IIF) test All dogs were immunised subcutaneously with 1 ml rabies vaccine (Rabisin , Merial, Italy). Forty days after the vaccine, administration blood samples were collected from the safena vein using 22 Gauge needles. Sera were obtained by centrifugation of blood for 30 min at 1000 g at 4 C, and sera were stored at 20 C until assayed for IIF according to the method of Buonavoglia et al. [3]. 2.4. Measurement of serum IFN- and IL-10 levels by ELISA Serum IFN- and IL-10 levels were measured in three samples of four animals each (two males and two females) selected among the animals showing signicant individual left-, right- or no-paw preferences in the behavioural test. The day before, and 5 and 10 days after the vaccine, administration blood samples were collected. Blood was allowed to clot for 2 h at room temperature before centrifuging for 30 min at 1000 g. Sera were stored at 20 C until assayed for canine IFN- and IL-10 by commercial kits (Quantikine ) purchased from R&D System, USA.

Fig. 3. Serum IFN- levels in right-, left- and ambi-preferent dogs (a) and a comparison of the results obtained the day before (T0), and 5 (T5) and 10 (T10) days after the vaccine administration (b) (group means with S.E.M. are shown).

3. Results 3.1. Anti-rabies antibody production The relative amounts of antibodies specic to rabies are shown in Fig. 2. The analysis of variance revealed a signicant main effect of paw preference in the serum levels of antirabies specic antibodies (F(2,9) = 7.176, P = 0.002). There was no main effect of sex (F(1,9) = 4.829, P = 1.000), nor any interaction between paw preference and sex (F(2,9) = 0.353, P = 0.705). Post hoc analyses (Fishers-protected LSD) revealed signicant differences between ambidextrous and left-preferent dogs (P = 0.0007), and between left-preferent and right-preferent dogs (P = 0.044) but not between ambipreferent and right-preferent dogs (P = 0.103). 3.2. Serum IFN- and IL-10 levels Serum levels of IFN- and IL-10 are shown in Figs. 3 and 4, respectively. The ANOVA revealed a signifFig. 4. Serum IL-10 levels in right-, left- and ambi-preferent dogs (a) and a comparison of the results obtained the day before (T0), and 5 (T5) and 10 (T10) days after the vaccine administration (b) (group means with S.E.M. are shown).

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icant main effect of paw preference in the serum levels of IFN- (F(2,9) = 7.622, P = 0.022); there was no main effect of sex (F(1,9) = 1.922, P = 0.215) and no signicant paw preference sex interaction (F(2,9) = 0.916, P = 0.449). Post hoc analyses (Fishers-protected LSD) revealed signicant differences between ambidextrous and left-preferent dogs (P = 0.009), and between left-preferent and right-preferent dogs (P = 0.030). No signicant effects of the time elapsed from the vaccine administration and blood samples collections were observed (main effect of time: F(2,9) = 2.410, P = 0.118), time paw preference interaction (F(4,9) = 0.755, P = 0.567). There were no signicant effects associated with serum IL-10 levels (Fig. 4; sex: F(1,9) = 0.989; paw preference: F(2,9) = 1.000); sex paw preference: F(2,9) = 0.452; time: F(2,9) = 1.213; time paw preference: F(2,9) = 1.109, P > 0.1 in all measures).

4. Discussion In the present study, the specic anti-rabies antibody production and the serum IL-10 and IFN- levels of mixed breed dogs were assayed for differential immune reactivities associated with paw preference. The main results can be summarised as follows. The titers of specic IgG anti-rabies antibodies after immunization were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. These ndings are in accordance with previous data that showed a lower number of -globulins in left-pawed than in right-pawed and ambidextrous dogs [17]. This is similar to a report by Kim et al. [11] that indicated a signicantly lower in vivo primary IgM and IgG anti-keyhole limpet hemocyanin antibody responses in mice with left-turning preference during spontaneous rotational behaviour in a circular cage. These results strengthened the hypothesis reported in mice that brain lateralisation could modulate the antibody production by the activity of sympathetic nerves, which are mainly distributed in the T cell area of the spleen [11,12]. No signicant treatment effect on the serum levels of IFN- was observed. This result could be related to the antigen employed in the present study. The IFN- production is mainly doing to Th-1 cells, which are activated by intracellular pathogens (viruses, bacteria, fungi and protozoa). In the present study, using an inactivated vaccine, the Th-2 response was electively induced with low or null IFN- production. In contrast, we have observed a signicant lateralisation effect on serum IFN- levels: left-pawed dogs displayed globally lower serum IFN- than right-pawed and ambidextrous dogs. This result appears to be in accordance with the observation that left-turners of untreated mice had lower serum IFN- levels than right-turners [11]. These ndings extend our previous results suggesting that brain laterality consistently modulates the immune system in an asymmetrical way and that this effect is present also before the immunization treatment. It

would be interesting in future work to assess the correlation between lateralisation and immune response as modulated by different levels of stress. The signicant correlation between IFN- and paw preference (with the latter being proved to be a reliable behavioural index of lateralisation [8,17], may indicate in this cytokine, a valid parameter to evaluate the relationship between brain laterality and immune system. The serum IL-10 levels seems to be not signicantly affected by behavioural lateralisation, in agreement with the ndings of Gao et al. [8] who reported that lateralisation does not inuence the plasma levels of IL-10 in mice selected for their paw preference, in response to LPS. However, concerning serum IL-10 levels, we observed a modest increment in left-pawed dogs compared with right-pawed and ambidextrous dogs. IL-10 was rst described as a Th-2 cytokine in mice that inhibited IFN-. On T cells, the initial observations of IL-10 inhibition of IFN- production has been successively suggested to be an indirect effect mediated by accessory cells [6,13]. Therefore, in left-pawed dogs, low IFN- serum may be related also to high serum levels of IL10 by the interaction of these two cytokines through lymphoid cells. Finally, an interesting aspect of these results that conrms our previous report [17] is that there was no hint of any interaction between sex and paw preference as to immune responses. This is intriguing considering that behavioural pawedness seems to exhibit different direction of bias in male and female dogs [17]. Immune response on the other hand reveals that ambidextrous and right-pawed dogs are consistently similar and different from left-pawed dogs, irrespective of sex. It could be therefore that gender effects in the direction of the asymmetry simply reect different modulations (likely by sex hormones) at the behavioural level of a basically similar neural asymmetry in the two sexes.

5. Conclusion In conclusion, the results clearly show that the direction of behavioural lateralisation inuences the specic antibody production, the serum levels of IFN- (but not those of IL-10) in response to rabies vaccine administration in dogs selected for their paw preference. The titers of anti-rabies antibodies were lower in leftpawed dogs than in right-pawed and ambidextrous dogs. Similarly, the IFN- serum levels were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. These ndings extend our previous results on the interaction of laterality and immune parameters in dogs, showing that the left-pawed group appeared to be consistently the different group, whereas immune responses tended to be similar in right-pawed and ambidextrous dogs. IL-10, as showed even by Dong et al. [5], seems to be an immune parameter not affected by brain lateralisation.

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