Toxocara canis larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS

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1 Parasitol Res (2011) 109: DOI /s y ORIGINAL PAPER Toxocara canis larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS Petra Kolbeková & David Větvička & Jan Svoboda & Karl Skírnisson & Markéta Leissová & Martin Syrůček & Helena Marečková & Libuše Kolářová Received: 24 January 2011 /Accepted: 25 March 2011 /Published online: 3 May 2011 # Springer-Verlag 2011 Abstract Using a small animal imaging system, migratory activity of Toxocara canis larvae stained by carboxyfluorescein succinimidyl ester (CFSE) was observed post primary infection (PPI) and post reinfection (PR) of BALB/c mice. Each infection was performed with 1,000 larvae per mouse. Primary infections were performed with labeled larvae, while for challenge infections the reinfecting P. Kolbeková : M. Leissová : L. Kolářová National Reference Laboratory for Tissue Helminthoses, General University Hospital in Prague, Prague, Czech Republic H. Marečková : L. Kolářová (*) Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Studničkova 7, Prague 2, Czech Republic libuse.kolarova@lf1.cuni.cz P. Kolbeková Department of Medical Microbiology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic D. Větvička : J. Svoboda Department of Immunology and Gnotobiology, Institute of Microbiology, Academy of Science, Prague, Czech Republic K. Skírnisson Institute of Experimental Pathology, University of Iceland, Keldur, Iceland M. Syrůček Department of Pathology, Na Homolce Hospital, Prague, Czech Republic larvae were stained by CFSE. The worm burden in mouse organs was determined during a period from 6 h to 21 days and 4 months PPI and PR. In comparison with primary infections that led to the first larvae appearance in the brain after 60 h, greatly accelerated migration of the parasites administered 3 weeks PPI to the CNS and eyes of challenged mice was noted in both organs the larvae appeared 6 h PR. In all challenged mice, reinfecting larvae prevailed in the resident parasite population. Preliminary experiments with Toxocara cati larvae also revealed early brain involvement in primarily infected mice. Staining of T. canis larvae by CFSE had no effect on the development of a humoral antibody response against T. canis excretory secretory antigens. In ELISA, elevated levels of specific IgG and IgG1 were noted on day 14 PPI and the levels of antibodies increased till the end of experiment. Reinfection induced an increase in the levels of both antibodies. In terms of optical density, IgG1 antibodies gave higher values in all sera examined. In ELISA for IgG antibodies, an increase in the avidity index of around 50% was detected 1 month PPI; higher-avidity antibodies were also detected in sera of reinfected animals. Abbreviations AI Avidity index BBB Blood brain barrier CFSE Carboxyfluorescein succinimidyl ester CNS Central nervous system DT Digestion technique OD Optical density PBS Phosphate-buffered saline PPI Post primary infection PR Post reinfection SAIS Small animal imaging system TES Toxocara canis excretory secretory antigens

2 1268 Parasitol Res (2011) 109: Introduction Roundworms of Toxocara canis and Toxocara cati are common gastrointestinal helminths of canids and felids all over the world and as a consequence of the widespread environmental contamination of their ova in host feces, other abnormal (paratenic) hosts including rodents, birds, and humans are also exposed to the parasites (Glickman and Schantz 1981). In paratenic hosts, the immature L3 larvae of the worms undergo somatic migration through the organs, however, they never mature. Invasion of larvae to a particular organ can lead to visceral, ocular, or neurological forms of the infection (Magnaval et al. 2001). Larvae of T. canis can be transmitted also from one paratenic host to another. Okoshi and Usui (1968) reported larval toxocarosis in mice inoculated with larvae originating from mouse or chickens and Pahari and Sasmal (1990) observed a similar situation in mice after infection with larvae originating from Japanese quails. Taira et al. (2004) successfully infected pigs with larvae originating from fresh swine or poultry viscera containing Toxocara larvae. In man, toxocarosis can be acquired by consumption of raw or undercooked meat or livers containing larvae (Nagakura et al. 1989; Kim et al. 2010). In humans, larval toxocarosis is one of the most common worldwide zoonotic helminthic infections (Schantz 1989). In comparison with the visceral form of the disease, human neurotoxocarosis is even now rarely diagnosed. The involvement of the host central nervous system (CNS) during infection can lead to dementia, eosinophilic meningitis, encephalitis or meningo-encephalitis, myelitis, cerebral vasculitis, arachnoiiditis, epilepsy, and spinal cord lesions. Manifestations of peripheral nervous system involvement include radiculitis, an effect on cranial nerves or musculoskeletal involvement (Finsterer and Auer 2007; Finsterer et al. 2010). Ocular toxocarosis develops when larvae enter the ocular chamber; it manifests as uveitis, endophthalmitis, papailitis, and retinal granuloma (Gillespie et al. 1993). It is suggested that young children are at especially high risk of infection because of their play habits and tendency to put their fingers in their mouths (Bächli et al. 2004). According to Uhlíková et al. (2002) ocular toxocarosis occur mainly in senior citizens. Moreira-Silva et al. (2004) suggested that neurotoxocarosis may occur at all stages and according to them there is no gender bias. Due to similarities seen between the progression of T. canis infection in mice and humans, the BALB/c mouse model for cerebral toxocarosis is frequently used for studies on behavior of larvae in paratenic hosts (Hamilton et al. 2006). It has been observed that larvae can invade the mouse CNS during the initial phase of infection; with cumulative infection doses and progress of infection, the probability of additional organ involvement increases (Sprent 1955; Dunsmore et al. 1983; Good et al. 2001; Hamilton et al. 2006; Camparoto et al. 2008). Neurotoxocarosis can be manifested by behavioral changes: the experimentally infected mice were less aggressive, less explorative, and showed some impairment in learning and memory (Cox and Holland 1998; Cox and Holland 2001a, b; Hamilton et al. 2006). The migratory behavior of T. canis and T. cati larvae is reported to be different in mice. According to many authors, T. cati larvae are rarely recovered from the mouse brain or eye (Sprent 1956; Prokopič and Figallová 1982; Havasiová-Reiterová et al. 1995). Toxocara larvae survive for a long period in paratenic hosts and antigenic stimulation leads to high levels of immunoglobulins which persist for a long period (Fenoy et al. 1992). Higher levels of antibodies can persist even after therapy of human infection (Uhlíková and Hübner 1983). Until now ELISA IgG with Toxocara excretory secretory antigens (TES) remains the method most used in laboratory diagnosis of the human disease, although it is not completely free of cross-reactivity with antigens of other helminths (Magnaval et al. 2001; Smith and Noordin 2006; Watthanakulpanich et al. 2008). In order to achieve higher sensitivity and specificity of ELISA reactions, IgG subclass antibodies were evaluated: Obwaller et al. (1998) reported that the predominant IgG subclass in human toxocariasis patients with visceral or asymtomaptic individuals was IgG1; in the study of Watthanakulpanich et al. (2008) IgG3 gave the best specificity when human sera were examined. Serological techniques leading to detection of IgG1 also showed higher sensitivity in comparison with IgG ELISAs performed with mouse sera (Fenoy et al. 2008). A correlation between the infective dose and the intensity of IgG or IgG1 humoral response was found by many authors (e.g., Kayes et al. 1985; Havasiová-Reiterová et al. 1995; Pinelli et al. 2007; Fenoy et al. 2008). Due to long-term persistence of elevated levels of antibodies in the infected paratenic hosts, it is important to distinguish between acute and chronic phases of infection. To differentiate between both phases of the infections, Hübner et al. (2001), Rychlicki (2004), and Dziemian et al. (2008) used a specific ELISA assay to measure levels of avidity of specific IgG against TES in human sera; low- or high-avidity antibodies indicated, respectively, recent or chronic infections. Fenoy et al. (2008) confirmed these findings in a T. canis/mouse model. There is little information on humoral responses in experimental mice infected by T. cati. Havasiová-Reiterová et al. (1995) reported that T. cati larvae elicited a lower overall humoral response as compared with T. canis larvae. Depending on the species of infected paratenic host, behavior of the parasites may vary and in this connection

3 Parasitol Res (2011) 109: the pathogenesis of infection may differ in different hosts (Kayes 2006). In our previous work (Kolbeková et al. 2011), we demonstrated a method of imaging viable T. canis larvae in mouse organs. Our data obtained from mice with a primary infection showed that staining of larvae by carboxyfluorescein succinimidyl ester (CFSE), followed by an epifluorescence detection method in a small animal imaging system (SAIS), allowed observation of the parasites in the organs of BALB/c mice till day 17 p.i. In the present study the migratory pattern of labeled T. canis larvae reinfecting mice of the same strain was investigated. Data on larval recovery in various organs obtained by SAIS were compared with a tissue digestion technique (DT). The evolution of IgG and IgG1 antibody response and the avidity of IgG antibodies against larvae stained by CFSE were determined as well. Although the study deals mainly with T. canis, some data obtained on experimental T. cati infections are also presented. Material and methods Toxocara canis The isolation and subsequent staining of T. canis larvae with CFSE (Invitrogen) was performed according methods described in our previous work (Kolbeková et al. 2011). Infection experiments Four groups of 3-month-old females Balb/c mice (AnLab, Ltd.) were intubated with T. canis larvae. In the first group, 18 mice were infected by a single dose of 1,000 CFSE-stained larvae; 18 mice from the second (control) group were infected by a single application of 1,000 non-stained larvae. In the third group, 22 mice were primarily infected by 1,000 non-stained larvae and 3 weeks later by 1,000 CFSE-labeled parasites; the fourth (control)groupconsistedof18miceinfectedinthesame manner, but all the administered larvae were not labeled. Two mice were left uninfected and served as a source for negative sera. Examinations by SAIS and DT were performed only on fresh organs isolated just after euthanizing of the animals by cervical dislocation. The stomach, intestine, liver, lungs, heart, kidney, spleen, brain, and eye were examined for the presence of larvae. The whole stomach, heart, kidney, spleen, and eyes were examined only by DT according to Kassai (1999); the material obtained from eyes was also examined under the fluorescent microscope. The liver and lungs were removed and divided into four equal parts; 1/4 was squashed slightly between two slides and examined by SAIS, the rest (3/4) by DT. The intestine was sliced into 12 parts; each fourth part was examined by SAIS, the rest of the organ by DT. The cerebellum was separated from the rest of the brain and hemispheres separated into left and right parts. The intact cerebellum and the hemispheres were examined by SAIS according to Kolbeková et al. (2011). Immediately after the examination the tissues were taken away from slides by washing with 1% pepsin and further observation was performed by DT. The organs of mice from the second and fourth control groups were examined only by DT. The worm burden was determined in the first and second groups of mice post primary infection (PPI) as well as in the third and fourth groups post reinfection (PR) at 6, 12, 24, 36, 48, 60, and 72 h p.i. and 7, 14, and 21 days p.i. In two challenged mice the distribution of larvae in the CNS was determined 4 months PR. For ethical reason, the number of experimental animals was kept low although this limited the statistical evaluation of data. Therefore, only results presented are the mean values and standard deviation. Histology Except for the eyes, small biopsies were isolated from each examined organ at any time point. At corresponding intervals, one mouse was enucleated at necropsy at the same time point as the examination for larval recovery by DT was performed and the whole eyes were examined histologically. The progress of pathological changes was evaluated on histological sections; tissues were fixed in 10% formaldehyde and stained by periodic-acid Schiff and hematoxylin eosin. Humoral antibody response Sera from mice experimentally infected by T. canis larvae were obtained after collection of peripheral blood from the tails of narcotized mice (Rometar and Narkomanon, Spofa). The first sample was obtained just before primary and challenge infections and additional serum samples were collected during the experimentation period. Two further mice with primary infections and two given a reinfection were kept alive for a longer period and sera from these were collected on days 14 and 21 and at 1, 3, and 4 months PPI, and 1, 3, and 4 months PR, respectively. Control negative sera are represented by samples obtained from animals just before the first inoculation of larvae as well as from the two uninfected mice. Observations on time course of the appearance of humoral response PPI and PR were performed by ELISA for IgG and IgG1 antibodies with TES prepared according to Watthanakulpanich et al. (2008).

4 1270 Parasitol Res (2011) 109: ELISA was carried essentially as described by de Savigny (1975) with some modification. The wells of microtiter plates (Plate NUNC Maxi Sorp) were treated with 100 μl of coating buffer (ph 9.6) containing TES (2.0 μg per well). The plates were incubated in a humidified chamber at 4 C overnight. Excess TES was removed by washing the wells three times with phosphatebuffered saline containing 0.05% Tween 20 (PBS/T, ph 7.2). One hundred microliters of sera diluted 1:200 in PBS/T were added to duplicate wells and the plates incubated at 37 C for 1 h. The wells were washed three times with PBS/T and 100 μl of peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich) diluted 1:10,000 or antimouse IgG1 (Caltag) diluted 1:2,000 in PBS/T were added to each well and incubated at 37 C for 1 h. After washing three time with PBS/T the reaction was developed by adding freshly prepared substrate solution (0.1 M C 6 H 5 Na 3 O 7. 2H 2 O, ph 5.0, with orthophenylendiamine, Sigma-Aldrich) containing 50 μl 30.0% H 2 O 2. The reaction was allowed to develop at RT for 15 min in the dark and was stopped by addition of 50 μl of 4 N H 2 SO 4. The absorbance was measured at 490 nm (Dynatech MR 50000) against the blank well. The cut off has been determined as double the mean optical density (OD) value given by sera obtained from mice before primary infection and from the uninfected mice. The evolution of the antibody response in terms of avidity has been performed by a similar ELISA for IgG according to Hübner et al. (2001). Each serum was investigated in quadruplicate (two sera with urea and two without) diluted 1:200 in PBS/T and incubated at 37 C for 1 h. After washing with PBS/T, two of the quadruplicated samples were drained from the wells and without washing 10 M urea in PBS was added immediately. The plates were incubated at 27 C for 20 min. After washing with PBS/T the anti-igg/peroxidase conjugate was added and the reaction continued as described above. Avidity index (AI) has been defined as the mean OD of urea-treated well/mean OD untreated wells 100 (Hübner et al. 2001; Fenoy et al. 2008). Values over 50% were ranked as antibody with high avidity (Fenoy et al. 2008). Toxocara cati The isolation, cultivation, and subsequent staining with CFSE of T. cati larvae were performed according to Kolbeková et al. (2011). In order to reach optimal degree of staining, several concentrations (1, 5, 10, and 40 μm) of CFSE in PBS were evaluated at various time intervals 15, 30, and 60 min, 1, 2, and 3 days. Six mice were intubated by non-stained T. cati larvae and the migration of the parasites has been evaluated by DT only. Results Toxocara canis The investigation of organs by SAIS showed that reinfecting T. canis larvae can be distinguished from the parasites causing previous infection. SAIS detected only CFSElabeled larvae during all monitored time points and showed persistence of the larval staining during the whole experiment, i.e., till 4 months (Fig. 2c). Observation under the fluorescent microscope showed that DT allows detection of both stained and non-stained larvae in the sediment obtained. Organ involvement by larvae In the visceral organs the T. canis larvae were detected by SAIS as well as by DT (Table 1). In comparison with mice with a primary infection, larvae appeared earlier in the liver, lungs, brain, and eyes of challenged animals. In the stomach of mice with primary and challenge infections, the maximum number of larvae was detected 12 h PPI and 6 h PR, respectively; solitary larva were detected even on day 7 PPI (Table 1). In the intestines of both groups of animals most larvae were found in animals 6 h PPI and PR; at 48 h PPI and PR, no larva was detected. The liver and lungs of mice with primary infections alone were invaded by larvae 6 h later than was the case in reinfected animals; i.e., involvement of the organs started 12 h PPI and 6 h PR, respectively. The maximum of larvae was observed in the liver 24 h PPI and 72 h PR, in the lungs 48 h PPI and PR. Solitary larvae were detected also in the heart: 0.5, 1, and 3.5 larvae per mouse at 36, 60, and 72 h PPI; in one reinfected mouse, one larva was detected 24 h PR. In challenged mice only, the kidney was invaded by few larvae 6 h, 12 h, and 7 days PR. No larva was detected in the spleens of any of the examined animals. The CNS involvement started at 60 h PPI (Table 2, Fig. 1), but in the challenged animals the brain became invaded by 6 h PR (Table 3, Fig. 1). As the infections progressed an increase in the number of brain larvae was noted in both groups of mice. Significantly more parasites were detected in the challenged animals at all time points; the highest number of larvae was recorded on day 21 PR. Four months PR, a decrease in the number of the brain larvae was noted. In the brains of reinfected animals larvae tended to accumulate mainly in the cerebral hemispheres (Table 3); they were not distributed randomly through the brain tissue and frequently were observed in aggregates (Fig. 3c). When the total number of brain larvae recovered by SAIS and DT in mice with primary infection or reinfection was compared, slightly higher values were noted by the

5 Parasitol Res (2011) 109: Table 1 The comparison of the mean number (±SD) of T. canis larvae in selected organs per one mouse detected by SAIS (in 1/4 of the organ) and DT (in 3/4 of the organ) post primary infection and reinfection; whole stomach examined only by DT Time p.i. No. mice Stomach Intestine Liver Lungs DT SAIS DT SAIS DT SAIS DT T AR A AR A AR A AR Primoinfection 6 h (±19.09) (±12.37) 83 (±21.21) h (±54.45) (±3.54) 27 (±9.90) (±2.12) 19.5 (±4.95) (±2.12) h 2 5 (±1.41) (±2.12) (±2.12) (±137.89) (±1.41) 4 (±1.41) h 2 3 (±2.83) (±0.71) (±4.24) 91.5 (±65.76) (±2.12) 4 (±5.66) h (±0.71) (±6.36) 74.5 (±7.78) (±0.71) 12.5 (±7.78) h ND (±6.36) 102 (±99) (±0.71) 5.5 (±6.36) h ND ND ND 144 (±83.44) ND 11 (±8.49) 7 d (±0.71) 0.1 ND ND 8 (±1.41) 44.5 (±17.68) (±2.12) 0.5 (±0.71) 0.2 Reinfection 6 h (±2.83) (±5.66) 75 (±5.66) (±2.12) (±0.71) h (±26.16) (±0.71) 12.5 (±2.12) (±2.12) 21.5 (±9.19) (±0.71) h 2 15 (±2.82) (±2.12) (±0.71) 84.5 (±26.16) (±0.71) h (±3.54) (±0.71) (±2.12) (±58.69) (±2.12) 5.5 (±4.95) h ND (±7.78) 70 (±2.83) (±4.95) h ND (±17.68) 87 (±8.49) 29 1 (±1.41) 2.5 (±0.71) h ND (±7.78) 154 (±67.88) (±1.41) 3.5 (±2.12) 1.2 7d ND (±2.12) 25.5 (±21.92) (±0.71) 1.5 (±2.12) d ND (±0.71) 16.5 (±2.12) (±0.71) d 2 ND ND (±5.66) T total number of larvae in the organ, A number of larvae in 3/4 of the organ, AR approximate rate in 1/4 of the organ, h hour, d day, m month Table 2 The mean number (±SD) of T. larvae detected by SAIS and DT in the brain during various phases post primary infection (PPI) Time PPI Primoinfection (PPI) No. mice SAIS DT 6h h h h h h (±2.12) 2 (±2.83) 72 h 2 ND 4.5 (±2.12) 7 d (±2.12) 10.5 (±2.12) 14 d 0 ND ND 21 d 0 ND ND 4 m 2 ND 75 (±8.48) h hour, d day, m month latter method (Tables 2 and 3, Fig. 1). Comparison of the data obtained by both methods in challenged animals revealed, however, that reinfecting larvae prevailed in the parasite population located in the brain at all time points PR. Ocular toxocarosis was observed mainly during challenge infections (Table 4). In comparison with a primary infection, eye involvement in challenged mice started earlier, i.e., at 60 h PPI and 6 h PR. And, more larvae arrived in the ocular chamber of challenged mice, in which involvement of both eyes was also frequently noted. No marked differences were recorded in the speed of migration of larvae to the organs in all control mice (data not shown). Histology No significant histopatological findings in organs from mice with either primary or challenged infection were observed. In the intestine histology revealed a weak infiltration by leukocytes in muscularis externa, sometimes accompanied by lesions like abscesses. In the liver inflammatory reactions containing mainly mononuclear

6 1272 Parasitol Res (2011) 109: Mean No. of larvae DT Reinfection SAIS cells were observed around the larvae. In some pulmonary areas disrupted alveoli with eosinophilic inflammation with polymorphonuclear cells were detected. Occasionally, perivascular infiltration, peribronchiolotis, and minimal alveolitis accompanied by infiltration with macrophages, eosinophils, and microhemorrhages were noted. Histology showed intra- as well as extravascular location in biopsies from the liver and lungs. With regard to the heart, light infiltration by lymphocytes was observed in the endocardium and sometimes, a weak infiltration of leukocytes was found. DT Primoinfection 4 m 14 d 72 h 48 h 24 h 6 h SAIS Fig. 1 The mean number of T. canis larvae in the mouse brain during various time intervals post primoinfection and reinfection detected by DT and SAIS; h hour, d day, m month Time p.i. In the brain (Fig. 2a), histopathology again did not reveal any severe tissue injuries. Some larvae were found between the cortex and white matter and occasionally under the pia mater. All recovered larvae were surrounded by tissue without significant immunological reactivity. However, occasionally leukostasis in the vessels was noted. In the cerebellum, we found only diminutive fresh abscesses. Gray matter of the organs was the most affected part and it was better-perfused than white matter. Due to retractional phenomenon it was impossible to evaluate intraor extravascular location of larvae in the brain tissue. Examination of the eyeballs (Fig. 2b) showed the development of weak perifocal edema starting by 12 h PR, but no significant additional pathological lesions were observed as the infection progressed. Humoral response The studies on the antibody response (Fig. 3) revealed an increase in the levels of specific IgG and IgG1 from day 14 till 4 months PPI. During reinfection the time course of antibody levels paralleled the situation observed in sera of mice with a primary infection alone. However, the mean OD of IgG and IgG1 reached higher values. In comparison with IgG, the mean OD of IgG1 in the ELISA was, respectively, approximately 1.8- and 1.4-fold greater in all sera of mice with primary infection and reinfection. The evolution of the IgG response in terms of avidity showed an increase in AI of around 50% since 1 month PPI (Fig. 3). In the sera of all challenged mice elevated values of AI were detected. Staining of larvae by CFSE did not influence the development of humoral responses in control mice in which the evolution of the antibody response showed a Table 3 The mean number (±SD) of T. larvae detected by SAIS and DT in the brain during various phases post reinfections (PR) Time PR No. mice Left part Right part Cerebellum Summary SAIS DT SAIS DT SAIS DT SAIS DT R (%) 6 h (±9.19) 33.5 (±13.44) 22.5 (±3.54) 31.5 (±7.78) 10.5 (±2.12) 24.5 (±3.54) 50.5 (±3.54) 89.5 (±24.75) h 2 18 (±7.07) 21 (±1.41) 29.5 (±2.12) 31.5 (±0.71) 8.5 (±2.12) 17 (±5.66) 56 (±2.83) 69.5 (±4.95) h 2 23 (±4.24) 24 (±2.83) 48 (±14.14) 50 (±12.73) 34 (±24.04) 40.5 (±16.26) 105 (±33.94) (±26.16) h 2 25 (±1.41) 35 (±5.66) 41.5 (±34.65) 48 (±28.28) 36.5 (±29.0) 38 (±29.70) 103 (±65.05) 121 (±52.3) h 2 17 (±19.8) 21 (±18.38) 14.5 (±0.71) 19.5 (±7.78) 11 (±4.24) 21 (±8.49) 42.5 (±24.75) 61.5 (±34.65) h 2 44 (±16.97) 48.5 (±19.09) 33.5 (±12.02) 37.5 (±6.36) 18 (±5.66) 26.5 (±0.71) 95.5 (±34.65) (±24.75) h (±13.44) 16 (±12.73) 13.5 (±7.78) 32 (±5.66) (±6.36) 34 (±5.66) 65.5 (±12.02) d 2 32 (±4.24) 43.5 (±3.54) 38.5 (±9.19) 49.5 (±10.61) 23 (±2.82) 23 (±1.41) 93.5 (±7.78) 116 (±8.49) d (±3.54) 51.5 (±6.36) 43 (±4.24) 51.5 (±4.95) 13.5 (±6.36) 33.5 (±4.95) 106 (±14.14) (±3.53) d (±4.95) ND 61.5 (±12.02) ND 30 (±2.82) ND 148 (±9.9) ND 4 m (±0.71) ND 12.5 (±7.78) ND 5 (±1.41) ND 30 (±8.49) ND R ratio of the reinfecting larvae, h hour, d day, m month

7 Parasitol Res (2011) 109: Table 4 The mean number (±SD) of T. canis larvae in the mouse eyes detected by DT and histology during various intervals post primary infection (PPI) and reinfection (PR) and the number of animals with unilateral (UNILAT) and bilateral (BILAT) infections Primoinfection Reinfection Time PPI No. mice No. larvae Time PR No. mice No. larvae Min Max No. FL UNILAT Σ mice BILAT Σ mice 6 h h (±2.08) (±0.58) h h (±2.31) (±1) h h (±2.31) (±1.15) h h (±0.58) (±1) h h 3 4 (±1.73) (±1.53) h (±0.71) 60 h (±1.15) (±1.53) h (±0.71) 72 h 3 2 (±1) (±1.53) d (±0.71) 7 d 2 2 (±2.83) (±2.12) d 0 ND 14 d 2 5 (±2.83) (±2.83) d 0 ND 21 d (±0.71) (±0.71) m 2 4 (±1.41) 4 m 0 ND ND ND No. FL the mean number (±SD) of fluorescent larvae detected in the sediment obtained by DT, h hour, d day, m month similar time course of the appearance of IgG and IgG1 and high-avidity antibodies (data not shown). T. cati Staining of the larvae by CFSE at any concentration led to early death of parasites and, therefore, six mice were infected by unlabeled T. cati larvae only. Using DT, one larva in the intestine, 71 larvae in the liver, 20 in the lungs, and one larva in the eye per animal were detected 60 h PPI; no larva was found in the stomach and brain. After 120 h PPI five, one, 16, and two larvae per mouse were recorded in the liver, lungs, brain, and heart, respectively; no larva was found in the stomach and eye. On day 8 PPI three, one, 21, and one larvae per mouse were found in the liver, heart, brain, and eye, respectively; no larva was detected in the stomach, intestine, and lungs. During time interval from h PPI, macroscopical observation showed large areas of hemorrhagic lung consolidation and histology revealed edema, congestion, and infiltration by neutrophils and eosinophils. Discussion The results presented here indicate that SAIS can be used to monitor migration of parasites infecting the hosts under different conditions. Our study showed that both SAIS and DT can give comparable results in studies of worm recovery in various organs. However, SAIS makes it possible to distinguish between parasites administered during various phases of infection as well as their long-term observation: the labeling persisted in T. canis larvae for 4 months. In addition, we observed that DT does not affect CFSE staining; under fluorescent microscope, labeled larvae were detected also in the sediment obtained. However, SAIS also revealed that CFSE is not optimal for labeling of each worm species. Whereas T. canis was relatively resistant to each concentration of CFSE (Kolbeková et al. 2011), T. cati larvae were more sensitive and most of them did not survive short-term stying within 1 h even when lower concentrations of the vital dye than recommended by a manufacturer protocol were used. In addition, staining of the larval intestine (Kolbeková et al. 2011) never reached the level observed in T. canis. The time course of the appearance of T. canis in mouse organs showed that larvae survived for a relatively long period in the host stomach (12 h PPI and PR) and it is still not clear whether these larvae are able to pass into the intestine and successfully migrate further to other organs. Nevertheless, the time course of the liver and lungs involvement PPI was similar to our previous data (Kolbeková et al. 2011) and those of others (Burren 1968, Abo-Shehada and Herbert 1984/1985, Hamilton et al. 2006). However, contrary to Pecinali et al. (2005) the number in pulmonary larvae never exceeded values detected in the liver in our mice. The number of larvae invading into the brain depends on the infection dose. It increases over the course of infection and the final larval count is influenced also by host immune status (Kayes and Oaks 1976, Abo-Shehada et al. 1991, Othman et al. 2010). With progress of the primary infection, we noted increasing accumulation of larvae in the mouse brain similarly as it was reported in many studies (Sprent 1955; Dunsmore et al. 1983; Bardón et al. 1994,

8 1274 Parasitol Res (2011) 109: Fig. 2 Toxocara canis larvae (arrows) a in the brain 48 h post reinfection, H&E; b in the eye retina 12 h post reinfection, H&E; c in clump formation 4 months PR, SAIS Fig. 3 Evolution of specific IgG and IgG1 response and IgG avidity in ELISA against TES during various phases of post primary infection (PPI) and reinfection (PR); h hour, d day, m month Optical density at 490nm Cut-off IgG1 10 Cut-off IgG Avidity index in % h 12 h 24 h 36 h 48 h 60 h 72 h 7d 14 d 21 d 1 m 3 m 4 m Time post-infection PPI IgG PR IgG PPI avidity IgG PPI IgG1 PR IgG1 PR avidity IgG 0

9 Parasitol Res (2011) 109: Skerrett and Holland 1997; Cox and Holland 2001a, b; Good et al. 2001; Hamilton et al. 2006; Camparoto et al. 2008; Othman et al. 2010). Also in mice reinfected 3 weeks PPI by T. canis, we noted an increase in larval count in the brain similarly as for Ollero et al. (2008) and Cox and Holland (2001a) who reinfected mice in weekly intervals. Similarly as in the brain, T. canis larvae arrived to the ocular chamber during primary as well as challenged infections. Compared with the situation PPI, the extensity and intensity of eye invasion was higher, and more animals suffered from bilateral eye involvement in reinfected mice. There is still no agreement regarding the relation between the parasitic load and the time of arrival of T. canis larvae to the eye. It is believed that ocular toxocarosis occurs in patients previously not sensitized by T. canis (Despommier 2003) because larvae are probably not trapped in the liver. In agreement with Kayes (2006), we confirmed that both visceral and ocular toxocarosis can develop during primary infection by massive parasite infectious dose, though similar clinical picture can develop also during reinfections. The time course of T. cati infections is in agreement with data of Cardillo et al. (2009), Olson (1976), Prokopič and Figallová (1982), and Ghafoor et al. (1984). We showed that the larvae can invade the brain and eye also during early phase of primary infection. The study can be criticized for a lack of statistical analysis, but satisfactory analysis was estimated to have required a minimum five mice necropsied each time and it was considered ethically unjustifiable, particularly in a situation in which heterogeneity of the behavior of Toxocara larvae in various paratenic hosts has been frequently reported (Hamilton et al. 2006). It has been documented that differences in the number of larvae recovered in paratenic hosts are due to a variety of factors such as species/strain of the animals used, size of the inoculum, duration of the infection, the host immune response, and laboratory method for larval recovery (Kayes et al. 1985; Abo-Shehada and Herbert 1989; Abo-Shehada et al. 1991; Epe et al. 1994; Cox and Holland 2001a; Hamilton et al. 2006). Last but not least, mice were examined in short time intervals and in any of them marked differences in the larval count were noted. In accordance with Sprent (1956), Burren (1968), Abo- Shehada et al. (1991), Dunsmore et al. (1983), Fan et al. (2003), and others, our results showed that larvae causing initial infection are able to migrate into the brain after successful passage through the liver when the immune response is weak to trap them. Similar factor was probably responsible for the rapid and massive invasion of the brain by reinfecting larvae that were intubated soon after initial infection, i.e., 3 weeks PPI. This view can be supported by the observation of Sugane and Oshima (1983) on athymic BALB/c-nu/+ mice reinfected by T. canis in various time intervals. The study showed that the number of larvae trapped in the liver increased with a prolongation of the interval between the primary and the secondary infection up to 6 weeks, but it did not increase when the interval was longer than 6 weeks. The reinfection performed at 6 weeks PPI caused rapid cellular infiltration around the larvae that were consequently trapped in the liver. Abo-Shehada et al. (1991) showed that the higher level of liver trapping is accompanied by a reduction of larval counts in the muscle and brain. The weak immune response on the day of reinfection was also confirmed serologically. Similarly to other reports (Kayes et al. 1985; Hrčková 2006; Bowman et al. 1987; Pinelli et al. 2007; Fenoy et al. 2008), the weak increase in the levels of specific IgG and IgG1 antibodies on day 14 PPI was noted, but significant increase in IgG was recorded since 1 month PPI. High-avidity IgG antibodies, which may signalize well-developed antigen antibody reactivity, were recorded 1 month PPI of mice, i.e., earlier than reported by Fenoy et al. (2008) who noted AI over 50% since day 60 PPI and suggested that it indicates chronic phase of infection. In comparison with adequate literary data (the same strain of mice and infection dose) we detected an increase in IgG1 response in time intervals similar to those reported by Pinelli et al. (2007) and Fenoy et al. (2008). Our data also confirmed higher sensitivity of ELISA assays when IgG subclasses are detected in human and mouse sera (Obwaller et al. 1998; Pinelli et al. 2007; Fenoy et al. 2008; Watthanakulpanich et al. 2008) Analogous to literary data (Sprent 1955; Burren 1968; Dunsmore et al. 1983; Fan et al. 2003), no severe pathologies were observed in the brain and eye in any of the infected animals. According to opinions of Smith and Noordin (2006) and Holland and Hamilton (2006), one of the possible mechanisms responsible for the lower degree of the brain injuries might be associated with minimization of neurological changes through less aggressive immune response by the CNS per se. Holland and Hamilton (2006) also supposed that T. canis larvae per se might be able to mimic a host s antigens and, therefore, they are able to escape host immune attacks. Considering the accelerated speed of T. canis larvae observed during early reinfection of mice in our study, we can also speculate that the weak immune response in sensitized mouse can stimulate the larvae to develop more effective immune evasion strategies compared with the infection of the naive host. Toxocara spp. larvae are reported to escape from the intestine and migrate through the blood stream via liver, right heart, lungs, and left heart to the brain. Usually, the lungs are reported as the first organ targeted by larvae escaping from the liver (Dunsmore et al. 1983; Magnaval et al. 2001; Akao 2006). However, similarly as Pecinali et al. (2005), we recorded that larvae arrived to the host lungs

10 1276 Parasitol Res (2011) 109: earlier than to the heart in some animals. In addition, we frequently observed the simultaneous presence of larvae in liver, lungs, brain, eye, and heart at the same time interval during early reinfection. Since histology showed intra- as well as extravascular locations in biopsies from liver and lungs, our findings indicate that, beside migration via the systemic circulation, the parasites migrated through tissues and the body cavity, as suggested by Abo-Shehada et al. (1984/1985), Bisseru (1969), and Burren (1968). Pecinali et al. (2005) supposed that this situation may occur due to larger fenestrations and/or a discontinuous capillary system in the host organs. Considering the eye involvement, contrary to Ollero et al. (2008), we detected T. cati larva in ocular chamber prior to the brain infection in one mouse with initial infection. Whereas, these authors suggest that ocular invasion starts from a previously colonized brain, our data show that larvae may enter also directly to the eye; the migration from arteria carotis interna to a. ophtalmica can be one of the possibilities. During invasion into the brain, Toxocara larvae have to cross the blood brain barrier (BBB), but the mechanisms which make it possible is unknown in detail. Generally, pathogens may cross the BBB trancellularly, paracellularly, and/or by means of infected phagocytes (for a review see Katchanov and Nawa 2010). Similarly, as for Angiostrongylus (Lee et al. 2006), it can be supposed that Toxocara spp. can cross BBB trans- or paracellularly. The BBB impairment is frequently associated with leukocytosis due to meningitis caused by various microbial agents (Stibbs and Curtis 1987, Lee et al. 2006, Brown et al. 2000). Although T. canis larvae could invade the area surrounding the plexus chorioideus, they did not elicit inflammatory cell infiltration or cerebral parenchymal injury in the brain (Epe et al. 1994, Fan et al. 2003, Liao et al. 2008). Therefore, in agreement with literary data (Dunsmore et al. 1983, Liao et al. 2008), leukocyte infiltration was not a feature of BBB impairment in our mice. Nevertheless, some studies showed that an increased larval count in the brain is associated with to biochemical and immunopathological changes that can be responsible for BBB impairment. The study of Hamilton et al. (2008) on a quantification of gene expression in the brain of susceptible (BALB/c) and resistant (NIH) mice to T. canis, the infection revealed increased expression of cytokines (IL-5, IL-10, and IFN-γ) and an inducible nitric oxide synthase (inos) as early as 3 days p.i., persisting till day 97 p.i.; this response was more pronounced in BALB/c mice. In agreement with Bandeira-Melo and Weller (2005), the authors suggest that the presence of IL-5 may result in the infiltration of eosinophils, followed by their subsequent degranulation accompanied by the release of toxic mediators that may cause dysfunction and destruction of surrounding cells. Regarding inos, it is induced in astrocytes and microglia by pathogenic components and pro-inflammatory cytokines such as TNF-α, IL-1, and IFNγ. Gargili et al. (2004) found that the enzyme has detrimental effects on the brain during infection with T. canis. They showed that mice treated with inos inhibitors displayed significantly less cerebral pathology than control mice; lower concentrations caused behavioral changes, higher amounts resulted in coma. Liao et al. (2008) reported that in mice with neurotoxocarosis, BBB injury was evidenced by enhanced glial fibrillary acidic protein (GFAP) expression (astrogliosis) in the brain from 4 to 8 weeks PPI. GFAP provides support and strength to astrocytes and its function may assist in maintaining the protective barrier which allows only certain substances to pass between blood vessels and the brain. In the presence of BBB injury, astrocytes may react more rapidly producing GFAP in response to the injury (Abbott et al. 2006). In mice infected by T. canis, Othman et al. (2010) recorded that progressive increase in the number of brain larvae was accompanied by increased expression of proinflammatory cytokines (TNF-α and IL-6) in a chronic stage of the infection (i.e., 5 16 weeks p.i.). TNF-α is secreted by many cell types such as glial cells and upregulated in many CNS disorders, including brain trauma and infections (Barone and Kilgore 2006). IL-6 has many functions, the primary of which is pro-inflammatory; it is produced by astrocytes, macrophages, microglia, neurons, and brain endothelial cells. IL-6 has both neuroprotective and neurotrophic functions and it is important in the activation of astrocytes and microglia, as well as in glial proliferation with subsequent gliosis (Swartz et al. 2001). Contrary to Hamilton et al. (2008), Liao et al. (2008), and Othman et al. (2010), we noted that the brain involvement started earlier. During the initial infection by T. canis, larvae arrived into the brain 60 h PPI, reinfecting larvae were able to occur in the organ as early as 6 h PR. We did not measure the levels of cytokines and other inflammatory markers. With respect to short time interval between primary and challenge infections and based on the studies of Swartz et al. (2001) and Barone and Kilgore (2006), we suppose that the increased BBB permeability in our experiments was dependent mainly on increased production of pro-inflammatory cytokines IL-6 and TNF that are produced by cells responsible for native immunity. It has been observed that the components of native immunity, e.g., Th17 lymphocytes, react more rapidly than those of adaptive immunity and contribute to the enhancement of BBB permeability (Neuwelt et al. 2008). Elucidating the mechanisms of immune response in the mouse brain provides important insights into the disease process and the consequences of human Toxocara infection which is usually acquired by ingestion of the parasite eggs.

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