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Complement membrane attack complex formation and infectivity of Trichinella spiralis and T. nativa in rats
Veterinary Parasitology 159 (2009) 263–267
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Complement membrane attack complex formation and infectivity of Trichinella spiralis and T. nativa in rats Anu Na¨reaho a,*, Seppo Saari a, Seppo Meri b, Antti Sukura a a b
Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, P.O. Box 66, University of Helsinki, 00014 Helsinki, Finland Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, University of Helsinki, 00014 Helsinki, Finland
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichinella Complement system Membrane attack complex Rat
Rats readily become infected with Trichinella spiralis but are more resistant to T. nativa. We infected complement factor C6-deficient (C6 ) rats and control (C6+) rats with T. spiralis and T. nativa to compare the effects of membrane attack complex on these parasites in vivo. The 2000 larvae infection dose per rat yielded 652 lpg (larvae per gram) in the C6 group and 608 lpg in the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were only 1.05 and 1.87 lpg. The difference between the Trichinella species was evident, but the infection intensity was unaffected by the C6 deficiency. When newborn larvae were incubated in C6-deficient and control rat sera for 24 h in vitro, no changes in viability were observed. Immunohistochemistry revealed that the musculature of crosssectioned adults and certain stichocytes bound human complement factors C3, C8 and C9, but not C1q. Interestingly, the outermost layer of the cuticle and the newborn larvae did not show any binding activity. Similar findings were obtained with immunofluorescence microscopy of intact newborn larvae. These results indicate that both T. spiralis and T. nativa have efficient mechanisms to protect themselves against complement attack. The difference in infectivity for rats between the two species, however, is not due to a differential resistance to complement membrane attack complex. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Trichinella’s life cycle consists of different developmental stages in different niches: muscle larvae inside muscle cells, adults in the small intestine, and newborn larvae (NBL) in the gut and circulation. Trichinella, especially the NBL, is thus exposed to different kinds of host defence mechanisms in its various living environments. Trichinella spiralis and T. nativa have different abilities to infect different host animals. Both species infect raccoon dogs equally well (Na¨reaho et al., 2000), but pigs and rats, for instance, are known to be fairly resistant to T. nativa infection (Kapel et al., 1998; Malakauskas et al.,
* Corresponding author. Tel.: +358 9 19157048. E-mail address: anu.nareaho@helsinki.fi (A. Na¨reaho). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.037
2001). Reasons behind the differences in infectivity are unknown. The complement system is a robust and fast innate immune defence mechanism against microbes and parasites invading the organ system (Walport, 2001). The classical pathway is activated mainly by antibodies, and the alternative pathway becomes activated without any prior exposure to the invader. Certain carbohydrates can activate the lectin pathway. Complement components act against pathogens by opsonising them for phagocytosis, launching inflammatory reactions, and also by perforating the cell membranes of susceptible targets (Rautemaa and Meri, 1999). The terminal complement proteins C5b–C9 are activated in a cascade to generate the membrane attack complex (MAC). The outcome is a membrane-destroying pore, which is composed mostly of C9 proteins. In C6 deficiency, the formation of MAC is prevented. Many
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pathogens can protect themselves against complement attack with several mechanisms (Finlay and McFadden, 2006). We analysed whether Trichinella, an invasive nematode, is resistant to complement attack and whether this resistance might play a role in the differential ability of T. spiralis and T. nativa to infect hosts. 2. Materials and methods 2.1. Study design Eighteen male PVG rats genetically deficient in complement factor C6 (C6 ) were obtained from Professor M. Daha, Leiden University Medical Centre, The Netherlands. Another 18 male PVG rats with a normal complement system (C6+) were from Harlan Netherland (Horst, The Netherlands). The phenotype of the C6 rat strain was confirmed from serum samples with a gel diffusion-based alternative pathway haemolytic complement kit (The Binding Site Ltd., Birmingham, UK) according to the manufacturer’s instructions. The rats were 12–19 weeks old at the beginning of the experiment. The normal-rat group was younger than the C6-deficient group for practical animal husbandry reasons. The animals’ weights ranged between 238 and 300 g at the study onset. In both the C6 and the C6+ group, six rats were infected with T. spiralis, six with T. nativa and six served as uninfected controls; thus 36 rats in total were included in the study. The T. spiralis strain (ISS 559) was originally from a pig, and T. nativa (ISS 558) from a raccoon dog. The species were identified at the Trichinella Reference Centre (Istituto Superiore di Sanita, Rome, Italy). Rats were infected per os with 2000 larvae. The condition of the rats was followed daily, and they were weighed weekly. The total duration of the experiment was 2 months, where after the rats were euthanized. Serum samples were collected before the infection and at 3 weeks p.i., as well as at the time of euthanasia. The Committee of Animal Experimentation of the Faculty of Veterinary Medicine approved the study protocol. 2.2. Newborn larvae Mice were infected with 100–200 muscle larvae of either T. spiralis or T. nativa. After 6 days, the mice were euthanized. The small intestine was removed immediately, cut longitudinally and rinsed with physiological saline. Adult, fertile female worms were collected from the intestine and cultured overnight in Dulbecco’s Modified Eagle’s medium at 10% CO2 and 37 8C (Marti and Murrell, 1986). The newborn larvae, released by adult female worms in culture, were washed with saline and exposed to undiluted normal and C6-deficient rat sera. NBL were also incubated in sera from T. spiralis, or T. nativa-infected rats with antibodies. Larval survival, i.e. movement, was monitored. The follow-up period consisted of 4 h with microscopic examination every hour, followed by examination again at 7 and 24 h. Approximately 100 NBL from at least three different females were exposed to each serum type. The sera were pooled from samples of several rats to eliminate individual variation.
2.3. ELISA The enzyme-linked immunosorbent assay (ELISA) for the detection of rat IgG anti-Trichinella antibodies used T. spiralis L1 E/S antigens (kindly provided by Dr. H.R. Gamble, National Research Council, Washington, DC, USA) and was performed as described elsewhere (Oivanen et al., 2005). 2.4. Immunohistochemistry and immunofluorescence microscopy For immunohistochemistry, Trichinella adults and NBL cultured overnight were washed with saline and collected into a test tube, and agarose was then poured on them. The agarose pellet with larvae in it was then embedded in paraffin and cut into 5 mm sections. Rat muscle samples with encapsulated Trichinella larvae were also sectioned. As controls, we used yeast cells (Saccharomyces cerevisiae), which strongly activate complement. The slides were incubated for 30 min in a 1:10 dilution of normal human serum, which acted as a complement source. We used four different primary antibodies for complement factors, each incubated for 30 min at 1:200 dilutions: rabbit anti-human C1q, rabbit anti-human C3 (Dako Cytomation, Glostrup, Denmark), goat anti-human C8 and goat anti-human C9 (Quidel, San Diego, CA, USA). To detect the binding of the primary antibody, we used a commercial kit according to the manufacturer’s instructions (LSAB + System-HRP, Daco Cytomation, Glostrup, Denmark). Control slides without normal human serum treatment and slides without primary antibody were included. To further study deposition of complement components on the surfaces of the living NBL and adults, we used indirect immunofluorescence microscopy. T. spiralis adults and NBL cultured overnight were collected into five 500 ml tubes: tube 1 was the primary sample, which underwent the entire procedure, while tubes 2–5 served as controls. Tube 2 was not treated with human serum, tube 3 was treated with inactivated human serum (56 8C, 40 min), tube 4 was not incubated with primary antibody and tube 5 was not incubated with secondary antibody. The same protocol was applied to tubes containing yeast (S. cerevisiae) as a positive control. All samples were washed four times with saline before beginning, and three times with 0.01 M PBS between the incubations. The first incubation was with 1:10 normal human serum for 30 min. To prevent non-specific binding reactions, all samples were treated for 5 min with 1% bovine serum albumin (BSA). Then the samples were incubated for 30 min with the same primary antibodies as in immunohistochemistry, in 1:200 dilutions. The next incubation was for 30 min with 1:100 fluorescein isothiocyanate (FITC)-conjugated secondary antibody (FITC Goat Anti-Rabbit IgG (H + L) Conjugate, or FITC Rabbit AntiGoat IgG (H + L) Conjugate, Zymed Laboratories Inc., San Francisco, CA, USA). After washing and centrifugation, the pellet from the bottom of the tube was pipetted on to a slide, mounted with Mowiol1 (Calbiochem, La Jolla, CA, USA) mounting medium, and observed with a Leica DM
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4000 B (Leica Microsystems, Wetzlar, Germany) microscope using a bright field filter and a filter specific for fluorescein. 2.5. Statistical analysis Differences between group means in the number of Trichinella recovered from muscle samples were compared using Student’s t-test. Comparisons were made both between the parasitic species and C6 and C6+ animals. The level of significance for all tests was set at p < 0.05.
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the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were 1.05 and 1.87 lpg. However, within each parasite species no significant differences in infection intensity between the C6 and C6+ groups were observed. Infected rats showed negligible clinical signs of trichinellosis, and only slower weight gain of the T. spiralisinfected animals was noticed in both C6+ and C6 groups, when compared to T. nativa-infected rats or controls. At the end of the experiment, mean weights in all experimental groups were within 300–350 g. 3.2. Antibody development in infected rats
3. Results 3.1. Clinical and laboratory analyses of infected rats Analyses of infection intensity (lpg) in rat muscles showed that T. spiralis was more infective than T. nativa in both C6 and C6+ groups (t-test: p < 0.01). The 2000 larvae infection dose per rat (equivalent to 7–8 larvae per gram, lpg) yielded 652 lpg in the C6 group and 608 lpg in
The levels of anti-Trichinella antibodies detected by ELISA increased faster during the first 3 weeks in the T. spiralis-infected groups than in the T. nativa groups, regardless of C6 status (data not shown). The higher infection intensity (lpg) in the T. spiralis group explains the titre difference at the 3-week sampling point between Trichinella species. The antibody levels reached plateau by the 8-week sampling point.
Fig. 1. Binding of complement components to Trichinella in sections of an adult female (T. nativa) with developing larvae inside (a, b, c, d) and mucle larvae (T. spiralis; e, f). Sections were incubated with human serum and analysed for binding of C1q (a), C3 (b), C8 (c) and C9 (d, e, f) using specific antibodies. In (a), no anti-C1q binding is seen. DL = developing larva, C = cuticle, U = uterus. In (b), the musculature and uterus (arrows) of adult are positively stained with antiC3, whereas the outermost layer of the cuticle as well as the unborn larvae are unstained. Similar but weaker staining intensity is seen with anti-C8 (c) and with anti-C9 (d). In (e), positively stained stichocytes are seen within T. spiralis muscle larvae. An enlargement of the insert is provided in (f). S = stichocyte. Scale bars = 20 mm (a, b, c, d, f) and 50 mm (e).
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3.3. Susceptibility of newborn larvae to complement lysis To study whether Trichinella larvae can be killed by serum complement, NBL of both T. spiralis and T. nativa were exposed to sera from C6+ and C6 rats and to sera from T. spiralis, and T. nativa-infected rats. The larvae survived and were moving in all tested sera throughout the 24 h follow-up. The presence or absence of antibodies in serum did not seem to affect the NBL. The adult females also survived the 24 h serum treatment. Thus, NBL and adults of both species of Trichinella were resistant to lytic complement attack. 3.4. Restricted complement component deposition on Trichinella To determine whether the resistance of Trichinella to complement lysis was due to lack of complement activation, we treated formalin-fixed and paraffin-embedded sections of larvae with serum and analysed binding of individual complement components immunohistochemically. None of the adult, newborn, or muscle Trichinella larvae bound complement factor C1q from human serum. Strongly positive staining for complement factor C3 was seen in certain stichocyte types in the stichosome of muscle larvae and in the musculature of adults. The outermost layer of the cuticle was always devoid of staining. Moreover, no binding by the unborn or newborn larvae was observed (Fig. 1). Factors C8 and C9 showed similar but weaker staining intensity than C3 (Fig. 1). No difference was observed between T. spiralis and T. nativa in binding of the complement factors. 3.5. Immunofluorescence microscopy analysis of complement component binding In the histological sections, sample fixation and processing could have interfered with the ability of the worms to bind complement components. Thus, we also tested fresh Trichinella worms for their ability to activate complement. Neither adults nor NBL showed any binding activity of C1q, C3, C8 or C9 from human serum (diluted 1:10) to the surface of the cuticle. Very strong diffuse binding of C3 was present on the surface of control samples containing serum-treated S. cerevisiae yeast. Punctate immunofluorescence was observed in the positive control samples where antibodies against C8 or C9 were used. No C1q binding was detected in any of the control samples. 4. Discussion Trichinella nematodes are invasive and inevitably exposed to complement attack as they traverse through tissues. Thus, the ability to evade complement is vital for their survival in the host. In this study, we examined whether differential resistance to complement by the two Trichinella species could at least partially explain their different infectivities to rats. However, both species of Trichinella were found to be resistant to killing by human as well as rat complement.
We investigated complement activation both in vivo and in vitro since evidence has emerged for T. spiralis that extrapolation of in vitro results may not be valid (Stankiewicz et al., 1989). All stages of Trichinella have been reported to be able to activate the complement system. Activation takes place mainly via the alternative pathway, the NBL being the most potent activators (Hong et al., 1992). Despite activation and consumption of complement, the larvae can evade complement attack and survive. In addition, human C3 has been shown to bind to the surface of muscle larvae of T. spiralis, whereas binding of C5 and C9 is minimal (Kennedy and Kuo, 1998). The lack of killing despite complement activation suggests that Trichinella larvae have efficient mechanisms to resist complement attack. The complement system culminates in the generation of MAC, which consists of components C5b–C9. Potentially, MAC would be capable of killing those cells of Trichinella nematodes that are not protected by a cuticle or a host cell membrane. We examined the role of MAC in trichinellosis by comparing infection in C6-deficient rats, in which the cascade to generate MAC is prevented, with infection in normal rats. The fact that no differences in larval survival between C6 and C6+ rat sera were observed indicates that larvae block complement attack prior to the C6 stage. Further studies are warranted to explore the precise molecular mechanism(s) of complement resistance of Trichinella. Within the muscle cell, Trichinella can avoid complement attack and other components of the immune system, but NBL live in the extracellular space and are exposed to the complement system. Killing by MAC is known to occur quite fast when the number of MACs on the cell membrane is high. We followed the NBL in human sera with and without Trichinella antibodies for several hours and did not observe any changes in their vitality. Thus, MAC seems to be unable to destroy NBL. However, Trichinella larvae have been shown to consume complement from serum (Hong et al., 1992). This suggests that either evasion of complement is an active process or Trichinella activates complement until the stage where evasion occurs. Immunohistochemistry and immunofluorescence microscopy showed that the complement factors C1q, C3, C5 and C9 do not bind on NBL. This is consistent with NBL surviving in the circulation. Factor C3 can become activated by any of the three complement pathways: classical, lectin or alternative. Here, it showed the strongest staining of all of the complement components. C3 binding was detected in the internal parts of Trichinella, especially in adults, but never in NBL. Interestingly, the outermost layer of the cuticle of Trichinella remained unstained. This was also observed in immunofluorescence analysis, where intact adults and NBL remained negative for C3 staining, but intense staining was seen on the surface of control yeast cells. A similar but weaker binding was noted with C8 and C9. Trichinella nematodes are covered by a cuticle that apparently forms a strong physical barrier against harmful insults, including complement attack. Shedding of the outer membrane during maturation of the larva from L1 to
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adult, or scaling of surface material can also liberate the worm from bound complement. It may also, for example, be able to capture complement inhibitors from the host. Previous studies have shown that Onchocerca volvulus microfilariae and Echinococcus granulosus bind the soluble alternative pathway inhibitor factor H to their surfaces (Meri et al., 2002). During growth Trichinella might acquire a protective shield containing CD59 protein, an inhibitor of MAC that prevents its polymerization. Possibly, Trichinella can, like schistosoma, synthesize complement inhibitors or enzymes that cleave complement factors (Jokiranta et al., 1995). The lack of staining for C3 and subsequent components, and the similar infectivities in C6 and C6+ rats suggest, however, that the main control occurs at the C3 level. In conclusion, T. spiralis and T. nativa seem to equally well control the complement system in vivo and in vitro. This ability obviously plays an important role in survival of adults, NBL and muscle larvae of Trichinella in the host. However, the complement evasion mechanisms utilized by Trichinella remain to be elucidated. This study showed that the difference in infectivity between T. spiralis and T. nativa in rats is not MAC-related.
Conflict of interest statement None of the authors has a financial or personal relationship with other people or organisations that could inappropriately influence or bias this article.
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References Finlay, B.B., McFadden, G., 2006. Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. Cell 124, 767–782. Hong, Y., Kim, C.W., Ghebrehiwet, B., 1992. Trichinella spiralis: activation of complement by infective larvae, adults, and newborn larvae. Exp. Parasitol. 74, 290–299. Jokiranta, T.S., Jokipii, L., Meri, S., 1995. Complement resistance of parasites. Scand. J. Immunol. 42, 9–20. Kapel, C.M., Webster, P., Lind, P., Pozio, E., Henriksen, S.A., Murrell, K.D., 1998. Trichinella spiralis, T. britovi, and T. nativa: infectivity, larval distribution in muscle, and antibody response after experimental infection of pigs. Parasitol. Res. 84, 264–271. Kennedy, M.W., Kuo, Y.M., 1998. The surfaces of the parasitic nematodes Trichinella spiralis and Toxocara canis differ in the binding of post-C3 components of human complement by the alternative pathway. Parasite Immunol. 10, 459–463. Malakauskas, A., Kapel, C.M., Webster, P., 2001. Infectivity, persistence and serological response of nine Trichinella genotypes in rats. Parasite 8, 216–222. Marti, H.P., Murrell, K.D., 1986. Trichinella spiralis: antifecundity and antinewborn larvae immunity in swine. Exp. Parasitol. 62, 370–375. Meri, T., Jokiranta, T.S., Hellwage, J., Bialonski, A., Zipfel, P.F., Meri, S., 2002. Onchocerca volvulus microfilariae avoid complement attack by direct binding of factor H. J. Infect. Dis. 185, 1786–1793. Nareaho, A., Sankari, S., Mikkonen, T., Oivanen, L., Sukura, A., 2000. Clinical features of experimental trichinellosis in the raccoon dog (Nyctereutes procyonoides). Vet. Parasitol. 91, 79–91. Oivanen, L., Na¨reaho, A., Jokela, S., Rikula, U., Gamble, R., Sukura, A., 2005. The prevalence of Trichinella infection in domestic dogs in Finland. Vet. Parasitol. 132, 125–129. Rautemaa, R., Meri, S., 1999. Complement resistance mechanisms of bacteria. Microbes Infect. 1, 785–794. Stankiewicz, M., Sokolska, G., Pilarczyk, A., Sadowska, D., Jeska, E.L., 1989. Lack of correlation between in vitro and in vivo activation of complement in mice by infective Trichinella spiralis larvae. J. Parasitol. 75, 647– 649. Walport, M.J., 2001. Complement. First of two parts. N. Engl. J. Med. 344, 1058–1066.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Complement membrane attack complex formation and infectivity of Trichinella spiralis and T. nativa in rats Anu Na¨reaho a,*, Seppo Saari a, Seppo Meri b, Antti Sukura a a b
Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, P.O. Box 66, University of Helsinki, 00014 Helsinki, Finland Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, University of Helsinki, 00014 Helsinki, Finland
A R T I C L E I N F O
A B S T R A C T
Keywords: Trichinella Complement system Membrane attack complex Rat
Rats readily become infected with Trichinella spiralis but are more resistant to T. nativa. We infected complement factor C6-deficient (C6 ) rats and control (C6+) rats with T. spiralis and T. nativa to compare the effects of membrane attack complex on these parasites in vivo. The 2000 larvae infection dose per rat yielded 652 lpg (larvae per gram) in the C6 group and 608 lpg in the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were only 1.05 and 1.87 lpg. The difference between the Trichinella species was evident, but the infection intensity was unaffected by the C6 deficiency. When newborn larvae were incubated in C6-deficient and control rat sera for 24 h in vitro, no changes in viability were observed. Immunohistochemistry revealed that the musculature of crosssectioned adults and certain stichocytes bound human complement factors C3, C8 and C9, but not C1q. Interestingly, the outermost layer of the cuticle and the newborn larvae did not show any binding activity. Similar findings were obtained with immunofluorescence microscopy of intact newborn larvae. These results indicate that both T. spiralis and T. nativa have efficient mechanisms to protect themselves against complement attack. The difference in infectivity for rats between the two species, however, is not due to a differential resistance to complement membrane attack complex. ß 2008 Elsevier B.V. All rights reserved.
1. Introduction Trichinella’s life cycle consists of different developmental stages in different niches: muscle larvae inside muscle cells, adults in the small intestine, and newborn larvae (NBL) in the gut and circulation. Trichinella, especially the NBL, is thus exposed to different kinds of host defence mechanisms in its various living environments. Trichinella spiralis and T. nativa have different abilities to infect different host animals. Both species infect raccoon dogs equally well (Na¨reaho et al., 2000), but pigs and rats, for instance, are known to be fairly resistant to T. nativa infection (Kapel et al., 1998; Malakauskas et al.,
* Corresponding author. Tel.: +358 9 19157048. E-mail address: anu.nareaho@helsinki.fi (A. Na¨reaho). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.10.037
2001). Reasons behind the differences in infectivity are unknown. The complement system is a robust and fast innate immune defence mechanism against microbes and parasites invading the organ system (Walport, 2001). The classical pathway is activated mainly by antibodies, and the alternative pathway becomes activated without any prior exposure to the invader. Certain carbohydrates can activate the lectin pathway. Complement components act against pathogens by opsonising them for phagocytosis, launching inflammatory reactions, and also by perforating the cell membranes of susceptible targets (Rautemaa and Meri, 1999). The terminal complement proteins C5b–C9 are activated in a cascade to generate the membrane attack complex (MAC). The outcome is a membrane-destroying pore, which is composed mostly of C9 proteins. In C6 deficiency, the formation of MAC is prevented. Many
264
A. Na¨reaho et al. / Veterinary Parasitology 159 (2009) 263–267
pathogens can protect themselves against complement attack with several mechanisms (Finlay and McFadden, 2006). We analysed whether Trichinella, an invasive nematode, is resistant to complement attack and whether this resistance might play a role in the differential ability of T. spiralis and T. nativa to infect hosts. 2. Materials and methods 2.1. Study design Eighteen male PVG rats genetically deficient in complement factor C6 (C6 ) were obtained from Professor M. Daha, Leiden University Medical Centre, The Netherlands. Another 18 male PVG rats with a normal complement system (C6+) were from Harlan Netherland (Horst, The Netherlands). The phenotype of the C6 rat strain was confirmed from serum samples with a gel diffusion-based alternative pathway haemolytic complement kit (The Binding Site Ltd., Birmingham, UK) according to the manufacturer’s instructions. The rats were 12–19 weeks old at the beginning of the experiment. The normal-rat group was younger than the C6-deficient group for practical animal husbandry reasons. The animals’ weights ranged between 238 and 300 g at the study onset. In both the C6 and the C6+ group, six rats were infected with T. spiralis, six with T. nativa and six served as uninfected controls; thus 36 rats in total were included in the study. The T. spiralis strain (ISS 559) was originally from a pig, and T. nativa (ISS 558) from a raccoon dog. The species were identified at the Trichinella Reference Centre (Istituto Superiore di Sanita, Rome, Italy). Rats were infected per os with 2000 larvae. The condition of the rats was followed daily, and they were weighed weekly. The total duration of the experiment was 2 months, where after the rats were euthanized. Serum samples were collected before the infection and at 3 weeks p.i., as well as at the time of euthanasia. The Committee of Animal Experimentation of the Faculty of Veterinary Medicine approved the study protocol. 2.2. Newborn larvae Mice were infected with 100–200 muscle larvae of either T. spiralis or T. nativa. After 6 days, the mice were euthanized. The small intestine was removed immediately, cut longitudinally and rinsed with physiological saline. Adult, fertile female worms were collected from the intestine and cultured overnight in Dulbecco’s Modified Eagle’s medium at 10% CO2 and 37 8C (Marti and Murrell, 1986). The newborn larvae, released by adult female worms in culture, were washed with saline and exposed to undiluted normal and C6-deficient rat sera. NBL were also incubated in sera from T. spiralis, or T. nativa-infected rats with antibodies. Larval survival, i.e. movement, was monitored. The follow-up period consisted of 4 h with microscopic examination every hour, followed by examination again at 7 and 24 h. Approximately 100 NBL from at least three different females were exposed to each serum type. The sera were pooled from samples of several rats to eliminate individual variation.
2.3. ELISA The enzyme-linked immunosorbent assay (ELISA) for the detection of rat IgG anti-Trichinella antibodies used T. spiralis L1 E/S antigens (kindly provided by Dr. H.R. Gamble, National Research Council, Washington, DC, USA) and was performed as described elsewhere (Oivanen et al., 2005). 2.4. Immunohistochemistry and immunofluorescence microscopy For immunohistochemistry, Trichinella adults and NBL cultured overnight were washed with saline and collected into a test tube, and agarose was then poured on them. The agarose pellet with larvae in it was then embedded in paraffin and cut into 5 mm sections. Rat muscle samples with encapsulated Trichinella larvae were also sectioned. As controls, we used yeast cells (Saccharomyces cerevisiae), which strongly activate complement. The slides were incubated for 30 min in a 1:10 dilution of normal human serum, which acted as a complement source. We used four different primary antibodies for complement factors, each incubated for 30 min at 1:200 dilutions: rabbit anti-human C1q, rabbit anti-human C3 (Dako Cytomation, Glostrup, Denmark), goat anti-human C8 and goat anti-human C9 (Quidel, San Diego, CA, USA). To detect the binding of the primary antibody, we used a commercial kit according to the manufacturer’s instructions (LSAB + System-HRP, Daco Cytomation, Glostrup, Denmark). Control slides without normal human serum treatment and slides without primary antibody were included. To further study deposition of complement components on the surfaces of the living NBL and adults, we used indirect immunofluorescence microscopy. T. spiralis adults and NBL cultured overnight were collected into five 500 ml tubes: tube 1 was the primary sample, which underwent the entire procedure, while tubes 2–5 served as controls. Tube 2 was not treated with human serum, tube 3 was treated with inactivated human serum (56 8C, 40 min), tube 4 was not incubated with primary antibody and tube 5 was not incubated with secondary antibody. The same protocol was applied to tubes containing yeast (S. cerevisiae) as a positive control. All samples were washed four times with saline before beginning, and three times with 0.01 M PBS between the incubations. The first incubation was with 1:10 normal human serum for 30 min. To prevent non-specific binding reactions, all samples were treated for 5 min with 1% bovine serum albumin (BSA). Then the samples were incubated for 30 min with the same primary antibodies as in immunohistochemistry, in 1:200 dilutions. The next incubation was for 30 min with 1:100 fluorescein isothiocyanate (FITC)-conjugated secondary antibody (FITC Goat Anti-Rabbit IgG (H + L) Conjugate, or FITC Rabbit AntiGoat IgG (H + L) Conjugate, Zymed Laboratories Inc., San Francisco, CA, USA). After washing and centrifugation, the pellet from the bottom of the tube was pipetted on to a slide, mounted with Mowiol1 (Calbiochem, La Jolla, CA, USA) mounting medium, and observed with a Leica DM
A. Na¨reaho et al. / Veterinary Parasitology 159 (2009) 263–267
4000 B (Leica Microsystems, Wetzlar, Germany) microscope using a bright field filter and a filter specific for fluorescein. 2.5. Statistical analysis Differences between group means in the number of Trichinella recovered from muscle samples were compared using Student’s t-test. Comparisons were made both between the parasitic species and C6 and C6+ animals. The level of significance for all tests was set at p < 0.05.
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the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were 1.05 and 1.87 lpg. However, within each parasite species no significant differences in infection intensity between the C6 and C6+ groups were observed. Infected rats showed negligible clinical signs of trichinellosis, and only slower weight gain of the T. spiralisinfected animals was noticed in both C6+ and C6 groups, when compared to T. nativa-infected rats or controls. At the end of the experiment, mean weights in all experimental groups were within 300–350 g. 3.2. Antibody development in infected rats
3. Results 3.1. Clinical and laboratory analyses of infected rats Analyses of infection intensity (lpg) in rat muscles showed that T. spiralis was more infective than T. nativa in both C6 and C6+ groups (t-test: p < 0.01). The 2000 larvae infection dose per rat (equivalent to 7–8 larvae per gram, lpg) yielded 652 lpg in the C6 group and 608 lpg in
The levels of anti-Trichinella antibodies detected by ELISA increased faster during the first 3 weeks in the T. spiralis-infected groups than in the T. nativa groups, regardless of C6 status (data not shown). The higher infection intensity (lpg) in the T. spiralis group explains the titre difference at the 3-week sampling point between Trichinella species. The antibody levels reached plateau by the 8-week sampling point.
Fig. 1. Binding of complement components to Trichinella in sections of an adult female (T. nativa) with developing larvae inside (a, b, c, d) and mucle larvae (T. spiralis; e, f). Sections were incubated with human serum and analysed for binding of C1q (a), C3 (b), C8 (c) and C9 (d, e, f) using specific antibodies. In (a), no anti-C1q binding is seen. DL = developing larva, C = cuticle, U = uterus. In (b), the musculature and uterus (arrows) of adult are positively stained with antiC3, whereas the outermost layer of the cuticle as well as the unborn larvae are unstained. Similar but weaker staining intensity is seen with anti-C8 (c) and with anti-C9 (d). In (e), positively stained stichocytes are seen within T. spiralis muscle larvae. An enlargement of the insert is provided in (f). S = stichocyte. Scale bars = 20 mm (a, b, c, d, f) and 50 mm (e).
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3.3. Susceptibility of newborn larvae to complement lysis To study whether Trichinella larvae can be killed by serum complement, NBL of both T. spiralis and T. nativa were exposed to sera from C6+ and C6 rats and to sera from T. spiralis, and T. nativa-infected rats. The larvae survived and were moving in all tested sera throughout the 24 h follow-up. The presence or absence of antibodies in serum did not seem to affect the NBL. The adult females also survived the 24 h serum treatment. Thus, NBL and adults of both species of Trichinella were resistant to lytic complement attack. 3.4. Restricted complement component deposition on Trichinella To determine whether the resistance of Trichinella to complement lysis was due to lack of complement activation, we treated formalin-fixed and paraffin-embedded sections of larvae with serum and analysed binding of individual complement components immunohistochemically. None of the adult, newborn, or muscle Trichinella larvae bound complement factor C1q from human serum. Strongly positive staining for complement factor C3 was seen in certain stichocyte types in the stichosome of muscle larvae and in the musculature of adults. The outermost layer of the cuticle was always devoid of staining. Moreover, no binding by the unborn or newborn larvae was observed (Fig. 1). Factors C8 and C9 showed similar but weaker staining intensity than C3 (Fig. 1). No difference was observed between T. spiralis and T. nativa in binding of the complement factors. 3.5. Immunofluorescence microscopy analysis of complement component binding In the histological sections, sample fixation and processing could have interfered with the ability of the worms to bind complement components. Thus, we also tested fresh Trichinella worms for their ability to activate complement. Neither adults nor NBL showed any binding activity of C1q, C3, C8 or C9 from human serum (diluted 1:10) to the surface of the cuticle. Very strong diffuse binding of C3 was present on the surface of control samples containing serum-treated S. cerevisiae yeast. Punctate immunofluorescence was observed in the positive control samples where antibodies against C8 or C9 were used. No C1q binding was detected in any of the control samples. 4. Discussion Trichinella nematodes are invasive and inevitably exposed to complement attack as they traverse through tissues. Thus, the ability to evade complement is vital for their survival in the host. In this study, we examined whether differential resistance to complement by the two Trichinella species could at least partially explain their different infectivities to rats. However, both species of Trichinella were found to be resistant to killing by human as well as rat complement.
We investigated complement activation both in vivo and in vitro since evidence has emerged for T. spiralis that extrapolation of in vitro results may not be valid (Stankiewicz et al., 1989). All stages of Trichinella have been reported to be able to activate the complement system. Activation takes place mainly via the alternative pathway, the NBL being the most potent activators (Hong et al., 1992). Despite activation and consumption of complement, the larvae can evade complement attack and survive. In addition, human C3 has been shown to bind to the surface of muscle larvae of T. spiralis, whereas binding of C5 and C9 is minimal (Kennedy and Kuo, 1998). The lack of killing despite complement activation suggests that Trichinella larvae have efficient mechanisms to resist complement attack. The complement system culminates in the generation of MAC, which consists of components C5b–C9. Potentially, MAC would be capable of killing those cells of Trichinella nematodes that are not protected by a cuticle or a host cell membrane. We examined the role of MAC in trichinellosis by comparing infection in C6-deficient rats, in which the cascade to generate MAC is prevented, with infection in normal rats. The fact that no differences in larval survival between C6 and C6+ rat sera were observed indicates that larvae block complement attack prior to the C6 stage. Further studies are warranted to explore the precise molecular mechanism(s) of complement resistance of Trichinella. Within the muscle cell, Trichinella can avoid complement attack and other components of the immune system, but NBL live in the extracellular space and are exposed to the complement system. Killing by MAC is known to occur quite fast when the number of MACs on the cell membrane is high. We followed the NBL in human sera with and without Trichinella antibodies for several hours and did not observe any changes in their vitality. Thus, MAC seems to be unable to destroy NBL. However, Trichinella larvae have been shown to consume complement from serum (Hong et al., 1992). This suggests that either evasion of complement is an active process or Trichinella activates complement until the stage where evasion occurs. Immunohistochemistry and immunofluorescence microscopy showed that the complement factors C1q, C3, C5 and C9 do not bind on NBL. This is consistent with NBL surviving in the circulation. Factor C3 can become activated by any of the three complement pathways: classical, lectin or alternative. Here, it showed the strongest staining of all of the complement components. C3 binding was detected in the internal parts of Trichinella, especially in adults, but never in NBL. Interestingly, the outermost layer of the cuticle of Trichinella remained unstained. This was also observed in immunofluorescence analysis, where intact adults and NBL remained negative for C3 staining, but intense staining was seen on the surface of control yeast cells. A similar but weaker binding was noted with C8 and C9. Trichinella nematodes are covered by a cuticle that apparently forms a strong physical barrier against harmful insults, including complement attack. Shedding of the outer membrane during maturation of the larva from L1 to
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adult, or scaling of surface material can also liberate the worm from bound complement. It may also, for example, be able to capture complement inhibitors from the host. Previous studies have shown that Onchocerca volvulus microfilariae and Echinococcus granulosus bind the soluble alternative pathway inhibitor factor H to their surfaces (Meri et al., 2002). During growth Trichinella might acquire a protective shield containing CD59 protein, an inhibitor of MAC that prevents its polymerization. Possibly, Trichinella can, like schistosoma, synthesize complement inhibitors or enzymes that cleave complement factors (Jokiranta et al., 1995). The lack of staining for C3 and subsequent components, and the similar infectivities in C6 and C6+ rats suggest, however, that the main control occurs at the C3 level. In conclusion, T. spiralis and T. nativa seem to equally well control the complement system in vivo and in vitro. This ability obviously plays an important role in survival of adults, NBL and muscle larvae of Trichinella in the host. However, the complement evasion mechanisms utilized by Trichinella remain to be elucidated. This study showed that the difference in infectivity between T. spiralis and T. nativa in rats is not MAC-related.
Conflict of interest statement None of the authors has a financial or personal relationship with other people or organisations that could inappropriately influence or bias this article.
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