Journal of Helminthology (2015) 89, 465–470 q Cambridge University Press 2014
doi:10.1017/S0022149X14000303
Behavioural changes and muscle strength in Rattus norvegicus experimentally infected with Toxocara cati and T. canis S.V. Santos1,2*, J.V.L. Moura2, S.A.Z. Lescano1, J.M. Castro3, M.C.S.A. Ribeiro2 and P.P. Chieffi1,2 1
Instituto de Medicina Tropical de Sa˜o Paulo - LIM 06, Avenida Doutor Eneias de Carvalho Aguiar, 450 - Cerqueira Cesar, Sa˜o Paulo, SP 05403-000, Brazil: 2Faculdade de Cieˆncias Me´dicas da Santa Casa de Sa˜o Paulo, Rua Doutor Cesa´rio Mota Jr., 61, Santa Cecı´lia, Sa˜o Paulo, SP 01221-020, Brazil: 3Prefeitura do Municı´pio de Sa˜o Paulo/SUVIS/Vila Maria/Vila Guilherme, Avenida Guilherme, 82, Vila Guilherme, Sa˜o Paulo, SP 02053-000, Brazil (Received 1 October 2013; Accepted 13 March 2014; First Published Online 11 April 2014) Abstract Toxocara canis and Toxocara cati are nematode parasites in dogs and cats, respectively, transmitted by ingestion of embryonated eggs, transmammary and transplacental (T. canis) routes and paratenic host predation. Many parasites use mechanisms that change the behaviour of their hosts to ensure continued transmission. Several researchers have demonstrated behavioural changes in mouse models as paratenic hosts for T. canis. However, there have been no studies on behavioural changes in laboratory rats (Rattus norvegicus) experimentally infected with T. cati. This study investigated behavioural changes and muscle strength in male and female rats experimentally infected with T. cati or T. canis in acute and chronic phases of infection. Regardless of sex, rats infected with T. cati showed a greater decrease in muscle strength 42 days post infection compared to rats infected with T. canis. However, behavioural changes were only observed in female rats infected with T. canis.
Introduction Parasitic alteration of host behaviour to facilitate dissemination has been reviewed by numerous researchers (Webster, 2007; Poulin, 2010; Holland & Hamilton, 2013). Some studies have reported evidence of behavioural changes in mice infected with Toxocara canis larvae related to the dose and parasite load in the brain (Cox & Holland, 2001a, b). However, before reaching the central nervous system, larvae pass through the musculature of the host, causing a decrease in muscle strength (Chieffi et al., 2009a). Toxocara canis and Toxocara cati are helminth parasites in dogs and cats, respectively. They are distributed
*E-mail: [email protected]
worldwide, occurring mainly in developing countries (Overgaauw, 1997; Chieffi et al., 2009b). Toxocara canis transmission in dogs occurs by ingestion of embryonated eggs found in the environment, predation of paratenic hosts, transmammary and/or transplacental routes, and by females swallowing young adults of the parasite after licking their offspring (Overgaauw, 1997; Queiroz & Chieffi, 2005). In feline T. cati transmission, transplacental transmission and swallowing young adults of the parasite have not been described (Sprent, 1956; Coati et al., 2004). Other animals, including humans, acting as paratenic hosts can become infected with these roundworms without developing adult worms. In this case, larvae migrate into the tissues and can remain in a latent stage and survive for a long time (Holland & Hamilton, 2013). However, there are some reports in the literature
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describing adults and children harbouring adult T. cati and T. canis specimens in their gut (Wiseman & Lovel, 1969; Eberhard & Alfano, 1998), likely due to accidental ingestion of young adults of the parasites. Donovick & Burright (1987) suggested that immunopathological reactions to T. canis may be both a cause of changes in behaviour and a symptom of infection. A history of T. canis exposure may alter reactions to subsequent exposures, which can change a broad spectrum of behaviours in mice, including reactivity to taste and exploration of the environment. Altered patterns of learning performance in mice and rats have been observed following infection with T. canis (Olson & Rose, 1966; Dolinsky et al., 1981). However, there are no reports concerning behavioural changes in laboratory rats (Rattus norvegicus) infected with T. cati. The present study evaluated behavioural changes and muscle strength in male and female rats experimentally infected with T. canis or T. cati in acute and chronic stages of infection.
Food and water were provided ad libitum. Cages were inspected daily and cleaned twice a week. Cages were housed in a room equipped with temperature control (198C/238C), an extractor fan and a device to control 12 h light/dark periods. A total of 50 6- to 8-week-old female R. norvegicus (Wistar) were divided into three groups, including two groups of 20 rats infected with 300 T. canis or T. cati eggs and a third group of 10 uninfected controls. All rats were marked with a yellow smear of saturated picric acid to differentiate among individuals in each cage. The same protocol was repeated for 50 male rats, to determine possible sex differences. The mean count of three microscope slides containing 50 ml of the egg culture solution was used to adjust the parasite dose to 300 eggs in 0.2 ml of a water solution for each rat (Lescano et al., 2004). Infection of rats was performed orally using a gavage needle. An equivalent amount of saline solution was similarly administered to rats in the control group.
Materials and methods
At 5, 15 and 42 days post infection (DPI), muscle strength in the forelimbs of female and male rats was evaluated using a grip strength meter (Ugo Basile, Comerio, Italy; cat. no. 47 105/47 106). In the apparatus, the rat is placed over a base plate in front of a T-shaped grasping bar fitted to a forced transducer connected to a peak amplifier. When pulled by the tail, the rat grasps at the bar until the pulling force overcomes its grip strength. When the rat loses its grip, the peak pull-force achieved by the forelimbs is shown on a liquid crystal display in grams and transformed to Newtons (N). Muscle strength was determined three times successively for each rat prior to and post infection. Body weights of all rats were also recorded.
Assessment of rat muscular strength Collection of adult worms and eggs of Toxocara Toxocara canis adults were recovered from naturally infected stray dogs captured by the Center for Zoonosis Control in Guarulhos, Sa˜o Paulo. Worms were placed in a glass receptacle containing saline solution and stored at 48C until use. Toxocara cati adults were obtained from three cats (approximately 2 months old) that had been donated to the Institute of Tropical Medicine in Sa˜o Paulo. Cats were maintained in individual 60 £ 60 £ 60 cm cages for 35 days. After this period, faecal samples were collected and examined by the sedimentation technique (De Carli, 2007). After confirming the presence of T. cati eggs, cats were treated with Endal Plusw tablets (20 mg praziquantel and 230 mg pyrantel pamoate) at a dose of one tablet per 4 kg weight. Adult worms were collected from cat faeces and female worms were dissected to obtain T. cati eggs. Toxocara canis and T. cati females were dissected in Petri dishes containing acidified water (pH 3), and uteri were removed and cut open to release eggs. The recovered eggs were concentrated by centrifugation at 1500 rpm for 5 min. The pellet containing the eggs was transferred to an Erlenmeyer flask containing approximately 200 ml of 2% formalin sealed with a hydrophobic cotton lid. The flask was placed in an incubator at 288C for approximately 30 days. Throughout the time period, it was manually agitated twice daily to ensure oxygenation of the eggs to promote development of larvae up to the third stage. After 30 days, which is the length of time required for third-stage larval formation, the eggs were washed three times in saline to remove the formalin solution and prepared for infection of rats. Maintenance and experimental infection of rats Six- to 8-week-old male and female R. norvegicus (Wistar) were obtained from the Main Animal Center at the Sa˜o Paulo University Medical School. Rats were separated into two groups of five specimens each and maintained in 49 £ 34 £ 16 cm polypropylene cages.
Assessment of rat behaviour and locomotion At 40 and 70 DPI, acute and chronic phases of infection, respectively, a 5-min elevated plus-maze (EPM) test was performed. The apparatus used in this experiment was made of plywood and consisted of two open (50 cm long £ 10 cm wide) and two enclosed (50 cm long £ 10 cm wide, with 40 cm high walls) arms connected by an open central area (10 £ 10 cm) arranged such that the two arms of each type were opposite each other and extended from a central platform elevated 50 cm above the ground (Handley & Mithani, 1984). Rats were initially placed on to the central platform of the maze, facing an open arm. Behaviour variables recorded included the number of entries into open (EOA) and closed arms (ECA) and the percentage of entries and time spent in open arms. The test apparatus was thoroughly cleaned with 5% ethanol between rats. A rat was considered to have entered an arm when all four limbs left the central area of the maze. Since this anxiety test reflects rats’ unconditioned aversion to heights and open spaces, the percentage of entries and time spent in open arms provide some measure of fear-induced inhibition of exploratory activity (Montgomery, 1955; Pellow et al., 1985, Pellow & File, 1986; Guaraldo et al., 2000; Carola et al., 2002). Spontaneous locomotor activity was measured using an animal activity cage (model 7430, Ugo Basile). The apparatus consisted of a transparent acrylic cage
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(35 £ 23 £ 20 cm) with a set of horizontal sensors to register locomotor activity and a set of vertical sensors to register standing activity (rearing). One day after the EPM test, each rat was placed alone inside the cage and locomotion and rearing were recorded for 5 min. During this period, immobility, grooming and faecal pellets were also recorded (Hall, 1934; Guaraldo et al., 2000; Lemos et al., 2010). Recovery and identification of larvae At the end of the experiment, all rats were euthanized to confirm infection, and the musculature and central nervous systems were digested with 0.5% HCl for 24 h at 378C. The supernatants were centrifuged for 2 min at 1500 rpm. Two millilitres of the sediments were collected and thoroughly mixed, and 0.1 ml samples were viewed under a light microscope for larval identification (Xi & Jin, 1998). Data analysis Data are expressed as the mean ^ SD. Analysis of variance for repeated measures followed by post hoc tests to adjust for multiple comparisons were used to compare muscle strength and body weight. Significant interactions resulted in use of analysis of variance for each time point (5, 15 and 42 DPI). The Kruskal– Wallis test was performed for EPM behaviour and locomotor activity tests. P values less than 0.05 were considered statistically significant, and all analyses were performed using SPSS v.17 (SPSS Inc., Chicago, Illinois, USA).
Results Muscle strength Muscle strength in female R. norvegicus was significantly decreased in infected animals at the three time points compared to controls (5 DPI: T. canis P ¼ 0.01 and T. cati P ¼ 0.03; 15 DPI: T. canis P ¼ 0.009 and T. cati P ¼ 0.01; and 42 DPI: T. canis P ¼ 0.00 and T. cati P ¼ 0.00). At 42 DPI, a significant difference between infected groups was also observed, with a greater decrease in the T. cati-infected group (T. canis versus T. cati P ¼ 0.03; table 1). In male R. norvegicus rats, there were significant decreases in muscle strength between infected and control groups (5 DPI: T. canis P ¼ 0.02 and T. cati P ¼ 0.01; 15 DPI: T. canis P ¼ 0.02 and
T. cati P ¼ 0.01; and 42 DPI: T. canis P ¼ 0.04 and T. cati P ¼ 0.01). No difference between infected groups was observed (table 1). No differences in body weight were verified between infected and control rats (table 1). Behaviour and locomotion Mean and standard deviations of entry frequency into open and closed arms, back of the open and closed arms, head dipping and time spent in the arms and centre by female R. norvegicus at 40 and 70 DPI are shown in table 2. Rattus norvegicus females infected with T. canis (34.6 ^ 26.3 s, P ¼ 0.028), but not T. cati (23.2 ^ 19.6 s, P ¼ 0.31), spent more time in the open arms than the control group (13.8 ^ 11.0 s). No statistically significant differences were observed between infected and control R. norvegicus males. Frequency of horizontal and vertical movements, time spent grooming, immobility and number of faecal pellets at 41 and 71 DPI were not statistically significant among groups.
Discussion Knowledge concerning the biology, epidemiology and physiopathology of infection of natural and paratenic hosts by T. cati is lacking compared with available information on T. canis (Fisher, 2003). This study improves the current understanding of several important aspects of host– parasite relationships established between T. cati and R. norvegicus, which is a common paratenic host for this ascarid. Few ecological relationships are as intimate as those between parasites and their hosts. Coexistence over time has allowed these organisms to create mutually beneficial adaptation mechanisms (Poulin, 1995). Behavioural changes could be considered adaptations that facilitate parasite transmission, primarily when prey – predation mechanisms are involved. Chieffi et al. (2010) observed that R. norvegicus infected with varying amounts of embryonated T. canis eggs (300 and 2000 eggs) showed a decrease in muscle strength in the forelimbs only 30 days after inoculation, although the number of eggs did not influence changes in strength. The authors affirmed that successive muscle strength measurements may result in more intense muscle fatigue in infected rats. In the present study, changes in muscle
Table 1. Relationship between muscle strength (Newtons) and body weight (g) (mean ^ SD) of female and male rats infected with T. cati or T. canis on days 5, 15 and 42 post infection; *significant differences with P , 0.05. Days post infection Parameter Female rats T. canis T. cati Control Male rats T. canis T. cati Control
5
15
42
Muscle strength
Body weight
Muscle strength
Body weight
Muscle strength
Body weight
348.8 ^ 100.4* 368.3 ^ 83.6* 471.0 ^ 58.5
252.4 ^ 34.8 256.5 ^ 24.1 253.1 ^ 20.6
319.1 ^ 98.7* 326.3 ^ 111.9* 461.5 ^ 94.6
244.3 ^ 13.7 253.3 ^ 13.0 264.5 ^ 23.2
216.2 ^ 74.4* 162.7 ^ 28.1* 356.0 ^ 43.2
275.5 ^ 16.8 271.2 ^ 15.0 277.5 ^ 22.6
114.7 ^ 30.8* 94.0 ^ 21.2* 249.1 ^ 21.9
197.2 ^ 19.1 199.8 ^ 24.4 188.3 ^ 12.6
157.1 ^ 30.2* 120.3 ^ 25.0* 285.9 ^ 47.9
236.4 ^ 25.1 249.3 ^ 26.7 237.2 ^ 21.4
402.5 ^ 73.5* 284.8 ^ 66.8* 561.5 ^ 56.5
362.4 ^ 34.2 347.0 ^ 47.2 365.0 ^ 28.0
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Table 2. Behaviour (mean ^ SD) of female R. norvegicus infected with T. cati or T. canis on days 40 and 70 post-infection in an elevated plus-maze; *significant differences at P , 0.05. Toxocara canis
Group Days post infection
40
Frequency in the maze (number of entries) Entries to open arms 2.1 ^ 2.1 Entries in closed arms 5.4 ^ 3.9 Back of open arms 0.2 ^ 0.7 Back of closed arms 4.4 ^ 2.7 Head dipping 0.2 ^ 0.4 Time spent (seconds) in the maze Open arms 34.6 ^ 26.3* Closed arms 239.1 ^ 46.8 Central platform 23.0 ^ 23.3
Toxocara cati
Control
70
40
70
40
70
1.9 ^ 1.4 3.3 ^ 2.4 0.6 ^ 0.8 2.7 ^ 1.9 0.1 ^ 0.3
1.0 ^ 1.1 5.6 ^ 2.8 0.1 ^ 0.4 5.0 ^ 2.3 0.1 ^ 0.3
1.0 ^ 0.9 3.5 ^ 2.5 0.3 ^ 0.4 2.6 ^ 1.7 0.0 ^ 0.0
0.5 ^ 0.7 3.3 ^ 1.9 0.5 ^ 0.7 3.2 ^ 1.9 0.1 ^ 0.3
1.8 ^ 1.6 3.3 ^ 2.7 0.3 ^ 0.5 3.0 ^ 2.8 0.0 ^ 0.0
98.4 ^ 99.1 178.1 ^ 101.9 23.4 ^ 26.4
23.2 ^ 19.6 255.8 ^ 28.8 21.1 ^ 16.7
54.3 ^ 69.2 224.4 ^ 71.1 21.2 ^ 24.3
13.8 ^ 11.0 268.6 ^ 19.7 19.5 ^ 15.5
37.7 ^ 30.3 238.1 ^ 48.6 24.1 ^ 19.7
strength in female and male R. norvegicus experimentally infected with T. cati or T. canis were evaluated 5, 15 and 42 days post infection. Both males and females infected by T. canis or T. cati had impaired muscle strength throughout the experimental period. However, females infected with T. cati showed greater loss of strength at 42 days post infection compared with the group infected with T. canis. Differences in muscle strength observed in rats infected with T. cati and T. canis are likely related to differences in larval migration patterns in rodents (Lescano et al., 2004; Santos et al., 2009) in that T. cati larvae show a predilection for muscles compared with T. canis larvae. In mice infected with T. canis, several studies have reported changes in exploratory behaviour and learning performance (Dolinsky et al., 1981; Burright et al., 1982; Hay et al., 1986; Donovick & Burright, 1987; Rodgers & Johnson, 1995; Ramos et al., 1997; Rodgers et al., 1997). In the present study, although a statistically significant difference was observed in entry frequency into open arms of the EPM by females infected with T. canis 40 DPI compared with the control group, no significant difference was observed at 70 DPI. Similar divergence has been reported by researchers working with rodents infected by Toxoplasma gondii. Hrda´ et al. (2000) also verified transient behavioural changes. Based on the results of the EPM test (table 2) and according to researchers who have studied anxiety levels using this type of apparatus (Handley & Mithani, 1984; Pellow & File, 1986), we suggest that infection by T. canis had an anxiolytic effect on female R. norvegicus. However, the influence of the oestrous cycle on this behaviour cannot be entirely discarded. Walf & Frye (2007) demonstrated that female mice in the dioestrus phase (low levels of oestradiol and progesterone) spend less time than males in open arms and spend more time than males during the proestrus phase (high levels of oestradiol and progesterone). However, discrepancies between authors in this regard have been noted (Mora et al., 1996; Marcondes et al., 2001). The present results show that spontaneous movement tests in the activity cage revealed no differences among the three groups of rats. This finding is in disagreement with that observed by Chieffi et al. (2010), who verified
significant differences in female R. norvegicus behaviour 30 days following infection with 2000 embryonated T. canis eggs, using the open-field test. These differences may be related to the number of eggs used to achieve infection and the infection period. Another factor that may have influenced results is differences in equipment. Behaviour in the open field was evaluated in a circular arena surrounded by a wooden wall (Chieffi et al., 2010) in which the floor of the apparatus was divided into 46 areas by circles and radial segments. In the present experiment, an activity cage was used to analyse behaviour in the open field. Unlike the arena, the activity cage consisted of transparent acrylic walls with no divisions marked on the floor. The absence of markings on the floor does not allow researchers to determine the frequency and length of time that the rat remained in the centre of the apparatus. The study of behavioural changes resulting from host– parasite relationships is extremely important because it increases understanding of the mechanisms parasites use to perpetuate their species. Knowledge of these mechanisms is necessary to develop measures for control and prevention of diseases related to parasitic infections.
Acknowledgements We thank the Center for Zoonosis Control of Guarulhos, Sa˜o Paulo, for providing Toxocara canis adult worms and BioMed Proofreading for English revision.
Financial support This work was supported by the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).
Conflict of interest None.
Ethical standards All procedures were performed strictly according to the guidelines for animal experimentation, as stipulated
Behavioural and muscular changes in Toxocara-infected rats
in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication Number 86 – 23, Bethesda, Maryland, USA). The experimental protocol was approved by the Research Ethics Committee on Animal Experiments of the Sa˜o Paulo Institute of Tropical Medicine (process no. 003/08).
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doi:10.1017/S0022149X14000303
Behavioural changes and muscle strength in Rattus norvegicus experimentally infected with Toxocara cati and T. canis S.V. Santos1,2*, J.V.L. Moura2, S.A.Z. Lescano1, J.M. Castro3, M.C.S.A. Ribeiro2 and P.P. Chieffi1,2 1
Instituto de Medicina Tropical de Sa˜o Paulo - LIM 06, Avenida Doutor Eneias de Carvalho Aguiar, 450 - Cerqueira Cesar, Sa˜o Paulo, SP 05403-000, Brazil: 2Faculdade de Cieˆncias Me´dicas da Santa Casa de Sa˜o Paulo, Rua Doutor Cesa´rio Mota Jr., 61, Santa Cecı´lia, Sa˜o Paulo, SP 01221-020, Brazil: 3Prefeitura do Municı´pio de Sa˜o Paulo/SUVIS/Vila Maria/Vila Guilherme, Avenida Guilherme, 82, Vila Guilherme, Sa˜o Paulo, SP 02053-000, Brazil (Received 1 October 2013; Accepted 13 March 2014; First Published Online 11 April 2014) Abstract Toxocara canis and Toxocara cati are nematode parasites in dogs and cats, respectively, transmitted by ingestion of embryonated eggs, transmammary and transplacental (T. canis) routes and paratenic host predation. Many parasites use mechanisms that change the behaviour of their hosts to ensure continued transmission. Several researchers have demonstrated behavioural changes in mouse models as paratenic hosts for T. canis. However, there have been no studies on behavioural changes in laboratory rats (Rattus norvegicus) experimentally infected with T. cati. This study investigated behavioural changes and muscle strength in male and female rats experimentally infected with T. cati or T. canis in acute and chronic phases of infection. Regardless of sex, rats infected with T. cati showed a greater decrease in muscle strength 42 days post infection compared to rats infected with T. canis. However, behavioural changes were only observed in female rats infected with T. canis.
Introduction Parasitic alteration of host behaviour to facilitate dissemination has been reviewed by numerous researchers (Webster, 2007; Poulin, 2010; Holland & Hamilton, 2013). Some studies have reported evidence of behavioural changes in mice infected with Toxocara canis larvae related to the dose and parasite load in the brain (Cox & Holland, 2001a, b). However, before reaching the central nervous system, larvae pass through the musculature of the host, causing a decrease in muscle strength (Chieffi et al., 2009a). Toxocara canis and Toxocara cati are helminth parasites in dogs and cats, respectively. They are distributed
*E-mail: [email protected]
worldwide, occurring mainly in developing countries (Overgaauw, 1997; Chieffi et al., 2009b). Toxocara canis transmission in dogs occurs by ingestion of embryonated eggs found in the environment, predation of paratenic hosts, transmammary and/or transplacental routes, and by females swallowing young adults of the parasite after licking their offspring (Overgaauw, 1997; Queiroz & Chieffi, 2005). In feline T. cati transmission, transplacental transmission and swallowing young adults of the parasite have not been described (Sprent, 1956; Coati et al., 2004). Other animals, including humans, acting as paratenic hosts can become infected with these roundworms without developing adult worms. In this case, larvae migrate into the tissues and can remain in a latent stage and survive for a long time (Holland & Hamilton, 2013). However, there are some reports in the literature
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describing adults and children harbouring adult T. cati and T. canis specimens in their gut (Wiseman & Lovel, 1969; Eberhard & Alfano, 1998), likely due to accidental ingestion of young adults of the parasites. Donovick & Burright (1987) suggested that immunopathological reactions to T. canis may be both a cause of changes in behaviour and a symptom of infection. A history of T. canis exposure may alter reactions to subsequent exposures, which can change a broad spectrum of behaviours in mice, including reactivity to taste and exploration of the environment. Altered patterns of learning performance in mice and rats have been observed following infection with T. canis (Olson & Rose, 1966; Dolinsky et al., 1981). However, there are no reports concerning behavioural changes in laboratory rats (Rattus norvegicus) infected with T. cati. The present study evaluated behavioural changes and muscle strength in male and female rats experimentally infected with T. canis or T. cati in acute and chronic stages of infection.
Food and water were provided ad libitum. Cages were inspected daily and cleaned twice a week. Cages were housed in a room equipped with temperature control (198C/238C), an extractor fan and a device to control 12 h light/dark periods. A total of 50 6- to 8-week-old female R. norvegicus (Wistar) were divided into three groups, including two groups of 20 rats infected with 300 T. canis or T. cati eggs and a third group of 10 uninfected controls. All rats were marked with a yellow smear of saturated picric acid to differentiate among individuals in each cage. The same protocol was repeated for 50 male rats, to determine possible sex differences. The mean count of three microscope slides containing 50 ml of the egg culture solution was used to adjust the parasite dose to 300 eggs in 0.2 ml of a water solution for each rat (Lescano et al., 2004). Infection of rats was performed orally using a gavage needle. An equivalent amount of saline solution was similarly administered to rats in the control group.
Materials and methods
At 5, 15 and 42 days post infection (DPI), muscle strength in the forelimbs of female and male rats was evaluated using a grip strength meter (Ugo Basile, Comerio, Italy; cat. no. 47 105/47 106). In the apparatus, the rat is placed over a base plate in front of a T-shaped grasping bar fitted to a forced transducer connected to a peak amplifier. When pulled by the tail, the rat grasps at the bar until the pulling force overcomes its grip strength. When the rat loses its grip, the peak pull-force achieved by the forelimbs is shown on a liquid crystal display in grams and transformed to Newtons (N). Muscle strength was determined three times successively for each rat prior to and post infection. Body weights of all rats were also recorded.
Assessment of rat muscular strength Collection of adult worms and eggs of Toxocara Toxocara canis adults were recovered from naturally infected stray dogs captured by the Center for Zoonosis Control in Guarulhos, Sa˜o Paulo. Worms were placed in a glass receptacle containing saline solution and stored at 48C until use. Toxocara cati adults were obtained from three cats (approximately 2 months old) that had been donated to the Institute of Tropical Medicine in Sa˜o Paulo. Cats were maintained in individual 60 £ 60 £ 60 cm cages for 35 days. After this period, faecal samples were collected and examined by the sedimentation technique (De Carli, 2007). After confirming the presence of T. cati eggs, cats were treated with Endal Plusw tablets (20 mg praziquantel and 230 mg pyrantel pamoate) at a dose of one tablet per 4 kg weight. Adult worms were collected from cat faeces and female worms were dissected to obtain T. cati eggs. Toxocara canis and T. cati females were dissected in Petri dishes containing acidified water (pH 3), and uteri were removed and cut open to release eggs. The recovered eggs were concentrated by centrifugation at 1500 rpm for 5 min. The pellet containing the eggs was transferred to an Erlenmeyer flask containing approximately 200 ml of 2% formalin sealed with a hydrophobic cotton lid. The flask was placed in an incubator at 288C for approximately 30 days. Throughout the time period, it was manually agitated twice daily to ensure oxygenation of the eggs to promote development of larvae up to the third stage. After 30 days, which is the length of time required for third-stage larval formation, the eggs were washed three times in saline to remove the formalin solution and prepared for infection of rats. Maintenance and experimental infection of rats Six- to 8-week-old male and female R. norvegicus (Wistar) were obtained from the Main Animal Center at the Sa˜o Paulo University Medical School. Rats were separated into two groups of five specimens each and maintained in 49 £ 34 £ 16 cm polypropylene cages.
Assessment of rat behaviour and locomotion At 40 and 70 DPI, acute and chronic phases of infection, respectively, a 5-min elevated plus-maze (EPM) test was performed. The apparatus used in this experiment was made of plywood and consisted of two open (50 cm long £ 10 cm wide) and two enclosed (50 cm long £ 10 cm wide, with 40 cm high walls) arms connected by an open central area (10 £ 10 cm) arranged such that the two arms of each type were opposite each other and extended from a central platform elevated 50 cm above the ground (Handley & Mithani, 1984). Rats were initially placed on to the central platform of the maze, facing an open arm. Behaviour variables recorded included the number of entries into open (EOA) and closed arms (ECA) and the percentage of entries and time spent in open arms. The test apparatus was thoroughly cleaned with 5% ethanol between rats. A rat was considered to have entered an arm when all four limbs left the central area of the maze. Since this anxiety test reflects rats’ unconditioned aversion to heights and open spaces, the percentage of entries and time spent in open arms provide some measure of fear-induced inhibition of exploratory activity (Montgomery, 1955; Pellow et al., 1985, Pellow & File, 1986; Guaraldo et al., 2000; Carola et al., 2002). Spontaneous locomotor activity was measured using an animal activity cage (model 7430, Ugo Basile). The apparatus consisted of a transparent acrylic cage
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(35 £ 23 £ 20 cm) with a set of horizontal sensors to register locomotor activity and a set of vertical sensors to register standing activity (rearing). One day after the EPM test, each rat was placed alone inside the cage and locomotion and rearing were recorded for 5 min. During this period, immobility, grooming and faecal pellets were also recorded (Hall, 1934; Guaraldo et al., 2000; Lemos et al., 2010). Recovery and identification of larvae At the end of the experiment, all rats were euthanized to confirm infection, and the musculature and central nervous systems were digested with 0.5% HCl for 24 h at 378C. The supernatants were centrifuged for 2 min at 1500 rpm. Two millilitres of the sediments were collected and thoroughly mixed, and 0.1 ml samples were viewed under a light microscope for larval identification (Xi & Jin, 1998). Data analysis Data are expressed as the mean ^ SD. Analysis of variance for repeated measures followed by post hoc tests to adjust for multiple comparisons were used to compare muscle strength and body weight. Significant interactions resulted in use of analysis of variance for each time point (5, 15 and 42 DPI). The Kruskal– Wallis test was performed for EPM behaviour and locomotor activity tests. P values less than 0.05 were considered statistically significant, and all analyses were performed using SPSS v.17 (SPSS Inc., Chicago, Illinois, USA).
Results Muscle strength Muscle strength in female R. norvegicus was significantly decreased in infected animals at the three time points compared to controls (5 DPI: T. canis P ¼ 0.01 and T. cati P ¼ 0.03; 15 DPI: T. canis P ¼ 0.009 and T. cati P ¼ 0.01; and 42 DPI: T. canis P ¼ 0.00 and T. cati P ¼ 0.00). At 42 DPI, a significant difference between infected groups was also observed, with a greater decrease in the T. cati-infected group (T. canis versus T. cati P ¼ 0.03; table 1). In male R. norvegicus rats, there were significant decreases in muscle strength between infected and control groups (5 DPI: T. canis P ¼ 0.02 and T. cati P ¼ 0.01; 15 DPI: T. canis P ¼ 0.02 and
T. cati P ¼ 0.01; and 42 DPI: T. canis P ¼ 0.04 and T. cati P ¼ 0.01). No difference between infected groups was observed (table 1). No differences in body weight were verified between infected and control rats (table 1). Behaviour and locomotion Mean and standard deviations of entry frequency into open and closed arms, back of the open and closed arms, head dipping and time spent in the arms and centre by female R. norvegicus at 40 and 70 DPI are shown in table 2. Rattus norvegicus females infected with T. canis (34.6 ^ 26.3 s, P ¼ 0.028), but not T. cati (23.2 ^ 19.6 s, P ¼ 0.31), spent more time in the open arms than the control group (13.8 ^ 11.0 s). No statistically significant differences were observed between infected and control R. norvegicus males. Frequency of horizontal and vertical movements, time spent grooming, immobility and number of faecal pellets at 41 and 71 DPI were not statistically significant among groups.
Discussion Knowledge concerning the biology, epidemiology and physiopathology of infection of natural and paratenic hosts by T. cati is lacking compared with available information on T. canis (Fisher, 2003). This study improves the current understanding of several important aspects of host– parasite relationships established between T. cati and R. norvegicus, which is a common paratenic host for this ascarid. Few ecological relationships are as intimate as those between parasites and their hosts. Coexistence over time has allowed these organisms to create mutually beneficial adaptation mechanisms (Poulin, 1995). Behavioural changes could be considered adaptations that facilitate parasite transmission, primarily when prey – predation mechanisms are involved. Chieffi et al. (2010) observed that R. norvegicus infected with varying amounts of embryonated T. canis eggs (300 and 2000 eggs) showed a decrease in muscle strength in the forelimbs only 30 days after inoculation, although the number of eggs did not influence changes in strength. The authors affirmed that successive muscle strength measurements may result in more intense muscle fatigue in infected rats. In the present study, changes in muscle
Table 1. Relationship between muscle strength (Newtons) and body weight (g) (mean ^ SD) of female and male rats infected with T. cati or T. canis on days 5, 15 and 42 post infection; *significant differences with P , 0.05. Days post infection Parameter Female rats T. canis T. cati Control Male rats T. canis T. cati Control
5
15
42
Muscle strength
Body weight
Muscle strength
Body weight
Muscle strength
Body weight
348.8 ^ 100.4* 368.3 ^ 83.6* 471.0 ^ 58.5
252.4 ^ 34.8 256.5 ^ 24.1 253.1 ^ 20.6
319.1 ^ 98.7* 326.3 ^ 111.9* 461.5 ^ 94.6
244.3 ^ 13.7 253.3 ^ 13.0 264.5 ^ 23.2
216.2 ^ 74.4* 162.7 ^ 28.1* 356.0 ^ 43.2
275.5 ^ 16.8 271.2 ^ 15.0 277.5 ^ 22.6
114.7 ^ 30.8* 94.0 ^ 21.2* 249.1 ^ 21.9
197.2 ^ 19.1 199.8 ^ 24.4 188.3 ^ 12.6
157.1 ^ 30.2* 120.3 ^ 25.0* 285.9 ^ 47.9
236.4 ^ 25.1 249.3 ^ 26.7 237.2 ^ 21.4
402.5 ^ 73.5* 284.8 ^ 66.8* 561.5 ^ 56.5
362.4 ^ 34.2 347.0 ^ 47.2 365.0 ^ 28.0
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Table 2. Behaviour (mean ^ SD) of female R. norvegicus infected with T. cati or T. canis on days 40 and 70 post-infection in an elevated plus-maze; *significant differences at P , 0.05. Toxocara canis
Group Days post infection
40
Frequency in the maze (number of entries) Entries to open arms 2.1 ^ 2.1 Entries in closed arms 5.4 ^ 3.9 Back of open arms 0.2 ^ 0.7 Back of closed arms 4.4 ^ 2.7 Head dipping 0.2 ^ 0.4 Time spent (seconds) in the maze Open arms 34.6 ^ 26.3* Closed arms 239.1 ^ 46.8 Central platform 23.0 ^ 23.3
Toxocara cati
Control
70
40
70
40
70
1.9 ^ 1.4 3.3 ^ 2.4 0.6 ^ 0.8 2.7 ^ 1.9 0.1 ^ 0.3
1.0 ^ 1.1 5.6 ^ 2.8 0.1 ^ 0.4 5.0 ^ 2.3 0.1 ^ 0.3
1.0 ^ 0.9 3.5 ^ 2.5 0.3 ^ 0.4 2.6 ^ 1.7 0.0 ^ 0.0
0.5 ^ 0.7 3.3 ^ 1.9 0.5 ^ 0.7 3.2 ^ 1.9 0.1 ^ 0.3
1.8 ^ 1.6 3.3 ^ 2.7 0.3 ^ 0.5 3.0 ^ 2.8 0.0 ^ 0.0
98.4 ^ 99.1 178.1 ^ 101.9 23.4 ^ 26.4
23.2 ^ 19.6 255.8 ^ 28.8 21.1 ^ 16.7
54.3 ^ 69.2 224.4 ^ 71.1 21.2 ^ 24.3
13.8 ^ 11.0 268.6 ^ 19.7 19.5 ^ 15.5
37.7 ^ 30.3 238.1 ^ 48.6 24.1 ^ 19.7
strength in female and male R. norvegicus experimentally infected with T. cati or T. canis were evaluated 5, 15 and 42 days post infection. Both males and females infected by T. canis or T. cati had impaired muscle strength throughout the experimental period. However, females infected with T. cati showed greater loss of strength at 42 days post infection compared with the group infected with T. canis. Differences in muscle strength observed in rats infected with T. cati and T. canis are likely related to differences in larval migration patterns in rodents (Lescano et al., 2004; Santos et al., 2009) in that T. cati larvae show a predilection for muscles compared with T. canis larvae. In mice infected with T. canis, several studies have reported changes in exploratory behaviour and learning performance (Dolinsky et al., 1981; Burright et al., 1982; Hay et al., 1986; Donovick & Burright, 1987; Rodgers & Johnson, 1995; Ramos et al., 1997; Rodgers et al., 1997). In the present study, although a statistically significant difference was observed in entry frequency into open arms of the EPM by females infected with T. canis 40 DPI compared with the control group, no significant difference was observed at 70 DPI. Similar divergence has been reported by researchers working with rodents infected by Toxoplasma gondii. Hrda´ et al. (2000) also verified transient behavioural changes. Based on the results of the EPM test (table 2) and according to researchers who have studied anxiety levels using this type of apparatus (Handley & Mithani, 1984; Pellow & File, 1986), we suggest that infection by T. canis had an anxiolytic effect on female R. norvegicus. However, the influence of the oestrous cycle on this behaviour cannot be entirely discarded. Walf & Frye (2007) demonstrated that female mice in the dioestrus phase (low levels of oestradiol and progesterone) spend less time than males in open arms and spend more time than males during the proestrus phase (high levels of oestradiol and progesterone). However, discrepancies between authors in this regard have been noted (Mora et al., 1996; Marcondes et al., 2001). The present results show that spontaneous movement tests in the activity cage revealed no differences among the three groups of rats. This finding is in disagreement with that observed by Chieffi et al. (2010), who verified
significant differences in female R. norvegicus behaviour 30 days following infection with 2000 embryonated T. canis eggs, using the open-field test. These differences may be related to the number of eggs used to achieve infection and the infection period. Another factor that may have influenced results is differences in equipment. Behaviour in the open field was evaluated in a circular arena surrounded by a wooden wall (Chieffi et al., 2010) in which the floor of the apparatus was divided into 46 areas by circles and radial segments. In the present experiment, an activity cage was used to analyse behaviour in the open field. Unlike the arena, the activity cage consisted of transparent acrylic walls with no divisions marked on the floor. The absence of markings on the floor does not allow researchers to determine the frequency and length of time that the rat remained in the centre of the apparatus. The study of behavioural changes resulting from host– parasite relationships is extremely important because it increases understanding of the mechanisms parasites use to perpetuate their species. Knowledge of these mechanisms is necessary to develop measures for control and prevention of diseases related to parasitic infections.
Acknowledgements We thank the Center for Zoonosis Control of Guarulhos, Sa˜o Paulo, for providing Toxocara canis adult worms and BioMed Proofreading for English revision.
Financial support This work was supported by the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).
Conflict of interest None.
Ethical standards All procedures were performed strictly according to the guidelines for animal experimentation, as stipulated
Behavioural and muscular changes in Toxocara-infected rats
in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication Number 86 – 23, Bethesda, Maryland, USA). The experimental protocol was approved by the Research Ethics Committee on Animal Experiments of the Sa˜o Paulo Institute of Tropical Medicine (process no. 003/08).
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