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Development and pathology of Fasciola hepatica in CCL3-deficient mice
Veterinary Parasitology 173 (2010) 147–151
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Development and pathology of Fasciola hepatica in CCL3-deficient mice R.C. De Paula a , G.D. Cassali b , D. Negrão-Corrêa a , M.P. Guimarães a,∗ a b
Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
a r t i c l e
i n f o
Article history: Received 26 May 2009 Received in revised form 10 June 2010 Accepted 10 June 2010 Keywords: Fasciola hepatica CCL3 Mice Experimental infection
a b s t r a c t Fasciola hepatica is a parasitic helminth that predominantly infects the liver and bile ducts of cattle and causes great losses of cattle production in the southern and southeastern regions of Brazil. The generation of liver lesions and the consequent inflammatory responses are intimately related to the migration of this parasite. The CC-group of chemokines plays a crucial role in the attraction of several cell types and in the recruitment of additional macrophages to an inflammatory focus in numerous diseases. In order to evaluate the role of CCL3 in the development of F. hepatica, we compared parasitological and pathological parameters in C57Bl/6J mice that were assigned to one of two experimental groups: the first group contained CCL3-producing mice (CCL3+/+ mice) and the other group contained mice that were genetically deficient in CCL3 production (CCL3−/− mice). The mortality rate in the CCL3 non-deficient group was higher than of the deficient animals. In most animals from both experimental groups, the necropsied animals contained hemorrhages in their abdominal cavities. In the genetically modified animals, the lesioned liver areas were less extensive and presented focal and sub-capsular lesions. This work demonstrates that the development of F. hepatica is not affected by the absence of CCL3. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fasciola hepatica Linnaeus (1758) are trematodes that inhabit the liver and bile ducts of their vertebrate hosts, including cattle, sheep, goats and horses (Queiroz et al., 2002). The economic losses that can be attributed to this fasciolosis are the result of a variety of factors, including the necessity to discard the liver and carcasses of infected animals in slaughterhouses, secondary bacterial infections, decreased dairy, meat and wool production and quality, weight loss, miscarriages, fertility decreases, delayed growth, and high mortality rates of the animals (Daemon and Serra-Freire, 1992; Oakley et al., 1979; Queiroz et al., 2002; Reid et al., 1972). The annual losses are estimated to reach U$3.2 billion worldwide (Yokananth et al., 2005). In addition to causing animal production losses, fasciolosis is
∗ Corresponding author. Tel.: +55 31 34092974; fax: +55 31 34092970. E-mail address: [email protected] (M.P. Guimarães). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.06.012
zoonotic; it infects humans, who may become accidental hosts (Müller et al., 1999). The definitive hosts acquire F. hepatica infection by ingesting metacercariae that excyst within the host gut lumen. Newly excysted juvenile flukes enter the intestinal epithelium, penetrate into the peritoneal cavity and then migrate through the liver before finally inhabiting the bile ducts. 2. Materials and methods 2.1. Parasites F. hepatica used in these experiments were isolated from naturally infected cattle, and the metacercariae were collected after experimental infection of Lymnaea columella according to Coelho et al. (2008). Briefly, parasite eggs were kept for 12–14 days at 26 ◦ C in Petri dishes that contained chlorine-free water to allow miracidia to develop. After this period, miracidium hatching was stimulated by artificial
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light and used to infect L. columella (3 miracidia/snail) that had been bred and maintained in the mollusk room of the helmintology laboratory—ICB-UFMG. After 60 days of F. hepatica infection, cercariae were collected from crushed L. columella and kept in tap water at room temperature until they were used for infection (maximum of 30 days). After this period, most metacercariae lose their viability (Müller et al., 1999). 2.2. Experimental animals
Table 1 Mortality rate in CCL3−/− and CCL3+/+ mice that were infected with 10 metacercariae of Fasciola hepatica. Weeks post-infection
Mortality rate (%)
1 2 3 4 5
CCL3+/+
CCL3−/−
0 0 0 64 73
0 0 0 10 60
The study was approved by the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (Protocol number 176/2006), and all procedures were carried out according to international guidelines. Fifty-two C57BL/6J mice were assigned to one of two experimental groups: Group 1—26 mice infected and Group 2—26 non-infected mice; 52 mice genetically deficient for the CCL3 gene (Cook et al., 1999) in the C57BL/6J background were also divided in the same manner (Groups 3 and 4). The mice were 10 ± 1 weeks old at the time of infection. Water and chow were given ad libitum.
Table 2 Number and location of parasites recovered during necropsy of C57Bl/6J mice that were infected with 10 metarcercariae of Fasciola hepatica (n = 52 per group).
2.3. Infection
in paraffin wax. Four-micrometer—thick sections were stained with hematoxylin–eosin (HE) and examined under an optical microscope.
Each mouse from Groups 1 and 3 was orally infected with 10 F. hepatica metacercariae, as previously described by Brady et al. (1999) and O’Neill et al. (2000). For the oral infection, the precise number of metacercariae was suspended in 100 l of distilled water, aspirated into a 1 ml syringe and then orally administered using a gavage needle. 2.4. Fecal exam Fecal examination of infected mice was carried out using the four sieves technique according to Ueno et al. (1975); a pool of feces was collected from each experimental group twice a week.
Parasite location
Groups CCL3+/+
CCL3−/−
Abdominal cavity Liver Bile duct
61 (78.2%) 16 (20.5%) 1 (1.3%)
45 (84.9%) 7 (13.2%) 1 (1.9%)
Total
78 (100%)
53 (100%)
2.7. Statistical analysis Results are expressed as means ± standard deviation of the mean (SDM), and comparisons between groups were carried out using Student’s t-test. Finally, the results were compared using an analysis of variance (one-way ANOVA) test, followed by the Newman–Keuls test for multiple comparisons. Values of p < 0.05 were considered to be statistically significant. 3. Results
2.5. Mortality rate and parasites
3.1. Mortality
The mortality rate in each experimental group was recorded. To recover parasite forms in different organs from each infected host and to evaluate macroscopic alterations caused by these parasites, the dead animals were necropsied. After opening the abdominal cavity of each animal longitudinally, they were checked for signs of hemorrhaging, and the cavity was then washed with 0.9% saline solution. The washing solution was recovered and analyzed using stereomicroscopy. Parasite forms that were found in the infected mice of each experimental group were transferred to a Petri dish that contained 0.9% saline solution, pressed between two glass dishes, fixed in formol at 10%, stained with carmin aceto-alumen, and then measured.
Infection with 10 metacercariae resulted in a high mortality rate for both CCL3−/− and CCL3+/+ mice. At 40 days post-infection (DPI), all of the infected animals had died. However, compared to CCL3−/− infected mice, F. hepaticainduced mortality began at an earlier stage in CCL3+/+ infected mice (Table 1).
2.6. Liver histopathology Fragments of the right lobes of livers from the necropsied animals were routinely processed and embedded
3.2. Parasitological parameters During the necropsy of infected animals, parasite forms were mostly found in the abdominal cavities of both CCL3−/− and CCL3+/+ mice. Flukes were also found in the liver and bile ducts (Table 2). The few flukes that were recovered from the bile duct were found in the wild type mice and in deficient mice that died at 23 and 26 DPI, respectively. No parasite flukes were recovered from other tissues. Although the flukes were found in the same locations in both the infected groups, the total number of flukes was always higher in the CCL3+/+ infected mice (Table 2).
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Fig. 1. Hepatic lesions induced by F. hepatica infection in CCL3 mice (A) and CCL3-deficient mice (B). (C) Histological liver section of a CCL3+/+ animal infected by F. hepatica stained by HE (10× increase). Extensive lesions caused by parasite migration are observed. Histological liver section of a CCL3−/− animal infected by Fasciola hepatica stained by HE (10× increase). Focal lesions (arrow), covering a small part of the damaged site.
The morphological characterization of flukes that were recovered from CCL3−/− and CCL3+/+ infected mice between 20 and 33 days of infection indicated that although they had a reproductive system, no eggs were found. Moreover, there was no statistical difference in the size of the flukes that were recovered from mice of each experimental group; their combined measurements were as follows: length = 198 (SDM = 44.3) and width = 96.9 (SDM = 13.7). The examination of feces that was performed for each of the groups during the infection period, yielded negative results.
3.3. Macroscopic alterations Several findings were common for both experimental groups during necropsy. The presence of blood in the abdominal cavity was concomitant with the finding of parasites in the cavity of both groups. An increased spleen size, suggestive of hypertrophy, was observed in the majority of the infected mice. The
spleen volume in CCL3-deficient mice was found to be slightly increased, whereas in the CCL3 non-deficient mice, there was a significant increase in the volume of this organ. In the CCL3−/− animal group, the liver size was slightly increased, whitish in color, contained focal sub-capsular lesions of size ranging from 1 to 5 mm long, around 1 to 3 mm wide and that were consistent with cuts that ran deep into the liver parenchyma (Fig. 1). The increase in the liver volume of the CCL3+/+ animals was more pronounced than in the CCL3−/− animals. In addition, most of the animals had caudal liver lobes that contained extensively damaged areas; these were possibly necro-hemorrhagic, and on occasions they extended almost across the entire lobe. In addition to the caudal lobe, large parts of the livers of some of the animals had the same type of lesion. Both groups exhibited alterations to their bile ducts, such as increased wall volume and thickness. However, such alterations were not different between the groups. A dark liquid inside the bile duct was also observed in both the CCL3−/− and the CCL3+/+ animals.
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3.4. Liver histopathology Staining and microscopic analysis of the liver sections confirmed a difference in the type of lesions that were found in the two groups. Compared to the CCL3+/+ animals, the area of damage in the CCL3−/− animals was less extensive. Also, in the same histological section, the focal characteristics of the lesions in the CCL3-deficient mice (Fig. 1) could be confirmed. In both groups, it was possible to visualize polymorphonuclear cell infiltrate surrounding the damaged sites, although this was more substantial in the CCL3+/+ mice. Areas of coagulative necrosis were also observed adjacent to the cellular infiltration areas; however, the areas of necrosis in the CCL3−/− mice were significantly smaller than those in the CCL3+/+ mice. 4. Discussion Our finding that infection with 10 F. hepatica metacercariae resulted in high rates of mortality, both in normal C57Bl/6J mice and in mice that were genetically deficient for the production of CCL3, is in agreement with the work of Dawes (1963c). In previous studies, there was also a high mortality rate in BALB/c mice that were infected with F. hepatica, only 5 weeks after infection (Marcet et al., 2002; Martinez-Fernandez et al., 2004; Lopéz-Aban et al., 2007). F. hepatica did not reach sexual maturity during infection in either normal C57Bl/6J mice or in animals that were genetically deficient in the production of CCL3. To the best of our knowledge, there are no reports in the literature on the elimination of F. hepatica eggs in the feces of either C57Bl/6J or BALB/c mice that were infected with the parasite. However, in the feces of AKRJ mice that were infected with three metacercariae per animal, Coelho et al. (2008) observed the presence of F. hepatica eggs at 27 days post-infection (DPI); this indicated that the parasite had reached sexual maturity in this strain of mouse. However, Hussein and Khalifa (2008) were unsuccessful in their attempts to infect white mice. In our study, the fact that many C57Bl/6J animals died before 27 DPI, partly explained the absence of mature parasites during the necropsy of these animals. However, even in mice that were necropsied after 29 DPI, sexually mature F. hepatica was not found. This suggested that there may be differences in the AKRJ strain that could conceivably support the development of the parasite (Bicalho et al., 2003). In R. rattus that was experimentally infected with F. hepatica, it was possible to confirm the development of adult parasites as well as the presence of viable eggs at 12 weeks post-infection (Valero et al., 2002). This indicated that compared to the R. rattus, mice may not be good hosts to use for studies on F. hepatica development. During necropsy of the infected C57Bl/6J mice, most parasites (81%) were recovered from the host’s abdominal cavity, moreover they were also found in the liver parenchyma and 1.5% of the total were located in the bile duct. However, as there were no statistically significant differences in the location of parasite between the mouse groups, it can also be concluded that CCL3 production by the host does not have a direct influence on either the migration process or on the development of the parasite.
The early death of the infected C57Bl/6J mice, combined with the physiological and anatomical characteristics of this host, may have denied most parasites access to the bile ducts and prevented them from attaining the adult phase. F. hepatica recovery in the abdominal cavity of the infected mice was associated with the frequent presence of intense abdominal hemorrhage. This observation, which is in agreement with Dawes (1963b), is related to the early death of C57Bl mice that were infected by F. hepatica associated with abdominal hemorrhage. Other authors also suggest that the acute hemorrhage phase of fasciolosis may be a consequence of the rupture of blood vessels during the migration of the parasite through the liver parenchyma and the constant movement in and out of the liver during the acute infection phase (Dawes, 1963a,b; Behm and Sangster, 1999). One can also speculate that the large number of parasites that were found in the abdominal cavity of the necropsied mice in this study may be a consequence of the parasite exiting the host’s liver parenchyma following the animal’s death. This can be explained by the fact that this organ is very rich in hydrolase-type enzymes, which trigger the autolysis process rapidly and turn the liver in an environment not suitable for the development of the parasite the liver into a barren ambient for the parasite (Pereira and Bogliolo, 2000). Despite the high mortality in infected C57Bl/6J mice, it was possible to demonstrate that the mortality induced by the parasite occurred much earlier in mice from the CCL3+/+ group than in those of the CCL3−/− group. This difference in mortality kinetics may be related to the number of parasites that were recovered from the mice; at 20 DPI, the number of parasites recovered from the CCL3+/+ mice was statistically larger (p < 0.05) than the amount recovered from the CCL3−/− mice. It was also noted that the extent of the lesions and cellular infiltration in the liver was much less in the CCL3−/− mice than in the CCL3+/+ mice. These results are similar to those found by Souza et al. (2005), in which they used the infection of CCL3+/+ and CCL3−/− mice with Schistosoma mansoni as their experimental model and verified that during the chronic phase of the infection, there were a statistically higher number of adult flatworms in the CCL3+/+ mice than in the CCL3−/− mice. In addition to the differential number of flatworms, it was also observed that the infection of CCL3−/− mice by S. mansoni resulted in a lesser degree of pathology; specifically, there was a smaller area of liver granulomes and collagen deposition. Thus, similar to the results obtained using the model of S. mansoni infection, the absence of CCL3 production during the infection by F. hepatica in the present study resulted in the recovery of a smaller number of parasites and the production of smaller liver lesions; these lesser effects resulted in a prolongation of the survival of the mice in this experimental group. According to Behm and Sangster (1999), the presence of liver fibrosis in the fasciolosis is due to the substitution of the inflammatory infiltrate that is present in the lesions with macrophages and fibroblasts. In the present study, there was less inflammatory infiltrate in the liver of CCL3−/− mice; this was a reflection of the fact that these cellular types are less abundant at these sites of injury and therefore there was a less extensive area of fibrosis in the
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CCL3−/− mice. Compared to the CCL3-defficient mice, mice that were capable of producing CCL3 had a higher mortality rate, a larger number of parasites recovered per animal, and more extensive liver lesions. To better elucidate the mechanisms that are involved in the formation of lesions via F. hepatica infection and the mechanisms that are responsible for parasitism in the vertebrate host, further studies are required. Acknowledgements We would like to thank Maria da Conceicao Caldeira and Hudson Andrade dos Santos for their technical assistance. References Behm, C.A., Sangster, N.C., 1999. Pathology, pathophysiology and clinical aspects. In: Dalton, X.X. (Ed.), Fasciolosis, 1a edic¸ão. CAB International, pp. 188–224. Bicalho, R.S., De Melo, A.L., Pereira, L.H., 2003. Schistosoma mansoni development in the peritoneal cavity of inbred AKR/J mice. Rev. Inst. Med. Trop. São Paulo 35, 411–416. Brady, M.T., O’Neill, S.M., Dalton, J.P., Mills, K.H.G., 1999. Fasciola hepatica suppresses a protective Th1 response against Bordetella pertussis. Infect. Immun. 67 (10), 5372–5378. Coelho, L.H.L., Guimaraes, M.P., Lima, W.S., 2008. Influence of shell size of Lymnaea columella on infectivity and development of Fasciola hepatica. J. Helminthol. 82, 77–80. Cook, D.N., Beck, M.A., Coffmann, T.M., Kirby, S.L., Sheridan, J.F., Pragnell, I.B., Smithies, O., 1999. Requeriment of MIP-1 alpha for an inflammatory response to viral infection. Science 286 (5447), 2098–2102. Daemon, E., Serra-Freire, N.M., 1992. Estudos da relac¸ão custo-benefício em parasitologia: uma proposta de análise. Parasitología al dia 16, 59–62. Dawes, B., 1963a. Hyperplasia of the duct biliar in fasciolosis and its relation to the problem of nutrition in the liver-fluke, Fasciola hepatica L. Parasitology 53, 123–133. Dawes, B., 1963b. The migration of juvenile forms of Fasciola hepatica L through the wall of the intestines in the mouse, with some observations on food and feeding. Parasitology 53, 109–122. Dawes, B., 1963c. Some observations of Fasciola hepatica L. during feeding operations in the hepatic parenchyma of the mouse, with notes on the nature of liver damage in this host. Parasitology 53, 135–143.
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Hussein, A.N., Khalifa, R.M., 2008. Experimental infections with Fasciola in snails, mice and rabbits. Parasitol. Res. 102 (6), 1165–1170. Lopéz-Aban, J., Casanueva, P., Nogal, J., Arias, M., Morrondo, P., Diez-Banos, P., Hillyer, G.V., Martinez-Fernandez, A.R., Muro, A., 2007. Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD. Vet. Parasitol. 145 (3–4), 287–296. Marcet, R., Diaz, A., Arteaga, E., Finlay, C.M., Sarracent, J., 2002. Passive protection against fasciolosis in mice by immunization with a monoclonal antibody (ES-78 MoAb). Parasite Immunol. 24, 103–108. Martinez-Fernandez, A.R., Nogal-Ruiz, J.J., Lopez-Aban, J., Ramajo, V., Oleaga, A., Manga-Gonzalez, Y., Hillyer, G.V., Muro, A., 2004. Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD. Vet. Parasitol. 15, 287–298. Müller, G., Berne, M.E.A., Raffi, L.L., Jesus, L.P., Paulsen, R.M.M., Sinkoc, A.L., 1999. Influência da temperatura na longevidade infectiva de metacercárias de Fasciola hepatica. Revista Brasileira Agrociências 5 (3), 164–165. O’Neill, S.M., Brady, M.T., Callanan, J.J., Mulcahy, G., Joyce, P., Mills, K.H.G., Dalton, J.P., 2000. Fasciola hepatica infection downregulates Th1 responses in mice. Parasite Immunol. 22, 147–155. Oakley, G.A., Owen, B., Knapp, N.H., 1979. Production effects of subclinical liver flukes infection in growing dairy heifers. Vet. Rec. 104 (22), 503–507. Pereira, F.E.L., Bogliolo, L., 2000. Inflamac¸ões. In: Patologia, 6aedic¸ão. Guanabara Koogan, pp. 113–148. Queiroz, V.S., Luz, E., Leite, L.C., Cirio, S.M., 2002. Fasciola hepatica (Trematoda, Fasciolidae) estudo epidemiológico nos municípios de Bocaiúva do Sul e Tunas do Paraná (Brasil). Acta Bio. Par. 31, 99–111. Reid, J.F., Doyle, J.J., Armour, J., Jennings, F.W., 1972. Fasciola hepatica infection in cattle. Vet. Rec. 90 (17), 486–487. Souza, A.L., Roffe, E., Pinho, V., Souza, D.G., Silva, A.F., Russo, R.C., Guabiraba, R., Pereira, C.A., Carvalho, F.M., Barsante, M.M., Correa-Oliveira, R., Fraga, L.A., Negrao-Correa, D., Teixeira, M.M., 2005. Potential role of the chemokine macrophage inflammatory protein 1␣ in human and experimental schistosomiasis. Infect. Immun. 73 (4), 2515– 2523. Ueno, H., Arandia, R., Morales, G., Medina, G., 1975. Fascioliasis of livestock and snail host for Fasciola in the altiplano region of Bolivia. Natl. Inst. Anim. Health Q. 15, 61–67. Valero, M.A., Panova, M., Comes, A.M., Fons, R., Mas-Coma, S., 2002. Patterns in size and shedding of Fasciola hepatica eggs by naturally and experimentally infected murid rodents. J. Parasitol. 88 (2), 308– 313. Yokananth, S., Ghosh, S., Gupta, S.C., Suresh, M.G., Saravanan, D., 2005. Characterization of specific and cross-reacting antigens of Fasciola gigantica by immunoblotting. Parasitol. Res. 97, 41–48.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Development and pathology of Fasciola hepatica in CCL3-deficient mice R.C. De Paula a , G.D. Cassali b , D. Negrão-Corrêa a , M.P. Guimarães a,∗ a b
Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
a r t i c l e
i n f o
Article history: Received 26 May 2009 Received in revised form 10 June 2010 Accepted 10 June 2010 Keywords: Fasciola hepatica CCL3 Mice Experimental infection
a b s t r a c t Fasciola hepatica is a parasitic helminth that predominantly infects the liver and bile ducts of cattle and causes great losses of cattle production in the southern and southeastern regions of Brazil. The generation of liver lesions and the consequent inflammatory responses are intimately related to the migration of this parasite. The CC-group of chemokines plays a crucial role in the attraction of several cell types and in the recruitment of additional macrophages to an inflammatory focus in numerous diseases. In order to evaluate the role of CCL3 in the development of F. hepatica, we compared parasitological and pathological parameters in C57Bl/6J mice that were assigned to one of two experimental groups: the first group contained CCL3-producing mice (CCL3+/+ mice) and the other group contained mice that were genetically deficient in CCL3 production (CCL3−/− mice). The mortality rate in the CCL3 non-deficient group was higher than of the deficient animals. In most animals from both experimental groups, the necropsied animals contained hemorrhages in their abdominal cavities. In the genetically modified animals, the lesioned liver areas were less extensive and presented focal and sub-capsular lesions. This work demonstrates that the development of F. hepatica is not affected by the absence of CCL3. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Fasciola hepatica Linnaeus (1758) are trematodes that inhabit the liver and bile ducts of their vertebrate hosts, including cattle, sheep, goats and horses (Queiroz et al., 2002). The economic losses that can be attributed to this fasciolosis are the result of a variety of factors, including the necessity to discard the liver and carcasses of infected animals in slaughterhouses, secondary bacterial infections, decreased dairy, meat and wool production and quality, weight loss, miscarriages, fertility decreases, delayed growth, and high mortality rates of the animals (Daemon and Serra-Freire, 1992; Oakley et al., 1979; Queiroz et al., 2002; Reid et al., 1972). The annual losses are estimated to reach U$3.2 billion worldwide (Yokananth et al., 2005). In addition to causing animal production losses, fasciolosis is
∗ Corresponding author. Tel.: +55 31 34092974; fax: +55 31 34092970. E-mail address: [email protected] (M.P. Guimarães). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.06.012
zoonotic; it infects humans, who may become accidental hosts (Müller et al., 1999). The definitive hosts acquire F. hepatica infection by ingesting metacercariae that excyst within the host gut lumen. Newly excysted juvenile flukes enter the intestinal epithelium, penetrate into the peritoneal cavity and then migrate through the liver before finally inhabiting the bile ducts. 2. Materials and methods 2.1. Parasites F. hepatica used in these experiments were isolated from naturally infected cattle, and the metacercariae were collected after experimental infection of Lymnaea columella according to Coelho et al. (2008). Briefly, parasite eggs were kept for 12–14 days at 26 ◦ C in Petri dishes that contained chlorine-free water to allow miracidia to develop. After this period, miracidium hatching was stimulated by artificial
148
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light and used to infect L. columella (3 miracidia/snail) that had been bred and maintained in the mollusk room of the helmintology laboratory—ICB-UFMG. After 60 days of F. hepatica infection, cercariae were collected from crushed L. columella and kept in tap water at room temperature until they were used for infection (maximum of 30 days). After this period, most metacercariae lose their viability (Müller et al., 1999). 2.2. Experimental animals
Table 1 Mortality rate in CCL3−/− and CCL3+/+ mice that were infected with 10 metacercariae of Fasciola hepatica. Weeks post-infection
Mortality rate (%)
1 2 3 4 5
CCL3+/+
CCL3−/−
0 0 0 64 73
0 0 0 10 60
The study was approved by the Ethical Committee for Animal Experimentation of the Universidade Federal de Minas Gerais (Protocol number 176/2006), and all procedures were carried out according to international guidelines. Fifty-two C57BL/6J mice were assigned to one of two experimental groups: Group 1—26 mice infected and Group 2—26 non-infected mice; 52 mice genetically deficient for the CCL3 gene (Cook et al., 1999) in the C57BL/6J background were also divided in the same manner (Groups 3 and 4). The mice were 10 ± 1 weeks old at the time of infection. Water and chow were given ad libitum.
Table 2 Number and location of parasites recovered during necropsy of C57Bl/6J mice that were infected with 10 metarcercariae of Fasciola hepatica (n = 52 per group).
2.3. Infection
in paraffin wax. Four-micrometer—thick sections were stained with hematoxylin–eosin (HE) and examined under an optical microscope.
Each mouse from Groups 1 and 3 was orally infected with 10 F. hepatica metacercariae, as previously described by Brady et al. (1999) and O’Neill et al. (2000). For the oral infection, the precise number of metacercariae was suspended in 100 l of distilled water, aspirated into a 1 ml syringe and then orally administered using a gavage needle. 2.4. Fecal exam Fecal examination of infected mice was carried out using the four sieves technique according to Ueno et al. (1975); a pool of feces was collected from each experimental group twice a week.
Parasite location
Groups CCL3+/+
CCL3−/−
Abdominal cavity Liver Bile duct
61 (78.2%) 16 (20.5%) 1 (1.3%)
45 (84.9%) 7 (13.2%) 1 (1.9%)
Total
78 (100%)
53 (100%)
2.7. Statistical analysis Results are expressed as means ± standard deviation of the mean (SDM), and comparisons between groups were carried out using Student’s t-test. Finally, the results were compared using an analysis of variance (one-way ANOVA) test, followed by the Newman–Keuls test for multiple comparisons. Values of p < 0.05 were considered to be statistically significant. 3. Results
2.5. Mortality rate and parasites
3.1. Mortality
The mortality rate in each experimental group was recorded. To recover parasite forms in different organs from each infected host and to evaluate macroscopic alterations caused by these parasites, the dead animals were necropsied. After opening the abdominal cavity of each animal longitudinally, they were checked for signs of hemorrhaging, and the cavity was then washed with 0.9% saline solution. The washing solution was recovered and analyzed using stereomicroscopy. Parasite forms that were found in the infected mice of each experimental group were transferred to a Petri dish that contained 0.9% saline solution, pressed between two glass dishes, fixed in formol at 10%, stained with carmin aceto-alumen, and then measured.
Infection with 10 metacercariae resulted in a high mortality rate for both CCL3−/− and CCL3+/+ mice. At 40 days post-infection (DPI), all of the infected animals had died. However, compared to CCL3−/− infected mice, F. hepaticainduced mortality began at an earlier stage in CCL3+/+ infected mice (Table 1).
2.6. Liver histopathology Fragments of the right lobes of livers from the necropsied animals were routinely processed and embedded
3.2. Parasitological parameters During the necropsy of infected animals, parasite forms were mostly found in the abdominal cavities of both CCL3−/− and CCL3+/+ mice. Flukes were also found in the liver and bile ducts (Table 2). The few flukes that were recovered from the bile duct were found in the wild type mice and in deficient mice that died at 23 and 26 DPI, respectively. No parasite flukes were recovered from other tissues. Although the flukes were found in the same locations in both the infected groups, the total number of flukes was always higher in the CCL3+/+ infected mice (Table 2).
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Fig. 1. Hepatic lesions induced by F. hepatica infection in CCL3 mice (A) and CCL3-deficient mice (B). (C) Histological liver section of a CCL3+/+ animal infected by F. hepatica stained by HE (10× increase). Extensive lesions caused by parasite migration are observed. Histological liver section of a CCL3−/− animal infected by Fasciola hepatica stained by HE (10× increase). Focal lesions (arrow), covering a small part of the damaged site.
The morphological characterization of flukes that were recovered from CCL3−/− and CCL3+/+ infected mice between 20 and 33 days of infection indicated that although they had a reproductive system, no eggs were found. Moreover, there was no statistical difference in the size of the flukes that were recovered from mice of each experimental group; their combined measurements were as follows: length = 198 (SDM = 44.3) and width = 96.9 (SDM = 13.7). The examination of feces that was performed for each of the groups during the infection period, yielded negative results.
3.3. Macroscopic alterations Several findings were common for both experimental groups during necropsy. The presence of blood in the abdominal cavity was concomitant with the finding of parasites in the cavity of both groups. An increased spleen size, suggestive of hypertrophy, was observed in the majority of the infected mice. The
spleen volume in CCL3-deficient mice was found to be slightly increased, whereas in the CCL3 non-deficient mice, there was a significant increase in the volume of this organ. In the CCL3−/− animal group, the liver size was slightly increased, whitish in color, contained focal sub-capsular lesions of size ranging from 1 to 5 mm long, around 1 to 3 mm wide and that were consistent with cuts that ran deep into the liver parenchyma (Fig. 1). The increase in the liver volume of the CCL3+/+ animals was more pronounced than in the CCL3−/− animals. In addition, most of the animals had caudal liver lobes that contained extensively damaged areas; these were possibly necro-hemorrhagic, and on occasions they extended almost across the entire lobe. In addition to the caudal lobe, large parts of the livers of some of the animals had the same type of lesion. Both groups exhibited alterations to their bile ducts, such as increased wall volume and thickness. However, such alterations were not different between the groups. A dark liquid inside the bile duct was also observed in both the CCL3−/− and the CCL3+/+ animals.
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3.4. Liver histopathology Staining and microscopic analysis of the liver sections confirmed a difference in the type of lesions that were found in the two groups. Compared to the CCL3+/+ animals, the area of damage in the CCL3−/− animals was less extensive. Also, in the same histological section, the focal characteristics of the lesions in the CCL3-deficient mice (Fig. 1) could be confirmed. In both groups, it was possible to visualize polymorphonuclear cell infiltrate surrounding the damaged sites, although this was more substantial in the CCL3+/+ mice. Areas of coagulative necrosis were also observed adjacent to the cellular infiltration areas; however, the areas of necrosis in the CCL3−/− mice were significantly smaller than those in the CCL3+/+ mice. 4. Discussion Our finding that infection with 10 F. hepatica metacercariae resulted in high rates of mortality, both in normal C57Bl/6J mice and in mice that were genetically deficient for the production of CCL3, is in agreement with the work of Dawes (1963c). In previous studies, there was also a high mortality rate in BALB/c mice that were infected with F. hepatica, only 5 weeks after infection (Marcet et al., 2002; Martinez-Fernandez et al., 2004; Lopéz-Aban et al., 2007). F. hepatica did not reach sexual maturity during infection in either normal C57Bl/6J mice or in animals that were genetically deficient in the production of CCL3. To the best of our knowledge, there are no reports in the literature on the elimination of F. hepatica eggs in the feces of either C57Bl/6J or BALB/c mice that were infected with the parasite. However, in the feces of AKRJ mice that were infected with three metacercariae per animal, Coelho et al. (2008) observed the presence of F. hepatica eggs at 27 days post-infection (DPI); this indicated that the parasite had reached sexual maturity in this strain of mouse. However, Hussein and Khalifa (2008) were unsuccessful in their attempts to infect white mice. In our study, the fact that many C57Bl/6J animals died before 27 DPI, partly explained the absence of mature parasites during the necropsy of these animals. However, even in mice that were necropsied after 29 DPI, sexually mature F. hepatica was not found. This suggested that there may be differences in the AKRJ strain that could conceivably support the development of the parasite (Bicalho et al., 2003). In R. rattus that was experimentally infected with F. hepatica, it was possible to confirm the development of adult parasites as well as the presence of viable eggs at 12 weeks post-infection (Valero et al., 2002). This indicated that compared to the R. rattus, mice may not be good hosts to use for studies on F. hepatica development. During necropsy of the infected C57Bl/6J mice, most parasites (81%) were recovered from the host’s abdominal cavity, moreover they were also found in the liver parenchyma and 1.5% of the total were located in the bile duct. However, as there were no statistically significant differences in the location of parasite between the mouse groups, it can also be concluded that CCL3 production by the host does not have a direct influence on either the migration process or on the development of the parasite.
The early death of the infected C57Bl/6J mice, combined with the physiological and anatomical characteristics of this host, may have denied most parasites access to the bile ducts and prevented them from attaining the adult phase. F. hepatica recovery in the abdominal cavity of the infected mice was associated with the frequent presence of intense abdominal hemorrhage. This observation, which is in agreement with Dawes (1963b), is related to the early death of C57Bl mice that were infected by F. hepatica associated with abdominal hemorrhage. Other authors also suggest that the acute hemorrhage phase of fasciolosis may be a consequence of the rupture of blood vessels during the migration of the parasite through the liver parenchyma and the constant movement in and out of the liver during the acute infection phase (Dawes, 1963a,b; Behm and Sangster, 1999). One can also speculate that the large number of parasites that were found in the abdominal cavity of the necropsied mice in this study may be a consequence of the parasite exiting the host’s liver parenchyma following the animal’s death. This can be explained by the fact that this organ is very rich in hydrolase-type enzymes, which trigger the autolysis process rapidly and turn the liver in an environment not suitable for the development of the parasite the liver into a barren ambient for the parasite (Pereira and Bogliolo, 2000). Despite the high mortality in infected C57Bl/6J mice, it was possible to demonstrate that the mortality induced by the parasite occurred much earlier in mice from the CCL3+/+ group than in those of the CCL3−/− group. This difference in mortality kinetics may be related to the number of parasites that were recovered from the mice; at 20 DPI, the number of parasites recovered from the CCL3+/+ mice was statistically larger (p < 0.05) than the amount recovered from the CCL3−/− mice. It was also noted that the extent of the lesions and cellular infiltration in the liver was much less in the CCL3−/− mice than in the CCL3+/+ mice. These results are similar to those found by Souza et al. (2005), in which they used the infection of CCL3+/+ and CCL3−/− mice with Schistosoma mansoni as their experimental model and verified that during the chronic phase of the infection, there were a statistically higher number of adult flatworms in the CCL3+/+ mice than in the CCL3−/− mice. In addition to the differential number of flatworms, it was also observed that the infection of CCL3−/− mice by S. mansoni resulted in a lesser degree of pathology; specifically, there was a smaller area of liver granulomes and collagen deposition. Thus, similar to the results obtained using the model of S. mansoni infection, the absence of CCL3 production during the infection by F. hepatica in the present study resulted in the recovery of a smaller number of parasites and the production of smaller liver lesions; these lesser effects resulted in a prolongation of the survival of the mice in this experimental group. According to Behm and Sangster (1999), the presence of liver fibrosis in the fasciolosis is due to the substitution of the inflammatory infiltrate that is present in the lesions with macrophages and fibroblasts. In the present study, there was less inflammatory infiltrate in the liver of CCL3−/− mice; this was a reflection of the fact that these cellular types are less abundant at these sites of injury and therefore there was a less extensive area of fibrosis in the
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CCL3−/− mice. Compared to the CCL3-defficient mice, mice that were capable of producing CCL3 had a higher mortality rate, a larger number of parasites recovered per animal, and more extensive liver lesions. To better elucidate the mechanisms that are involved in the formation of lesions via F. hepatica infection and the mechanisms that are responsible for parasitism in the vertebrate host, further studies are required. Acknowledgements We would like to thank Maria da Conceicao Caldeira and Hudson Andrade dos Santos for their technical assistance. References Behm, C.A., Sangster, N.C., 1999. Pathology, pathophysiology and clinical aspects. In: Dalton, X.X. (Ed.), Fasciolosis, 1a edic¸ão. CAB International, pp. 188–224. Bicalho, R.S., De Melo, A.L., Pereira, L.H., 2003. Schistosoma mansoni development in the peritoneal cavity of inbred AKR/J mice. Rev. Inst. Med. Trop. São Paulo 35, 411–416. Brady, M.T., O’Neill, S.M., Dalton, J.P., Mills, K.H.G., 1999. Fasciola hepatica suppresses a protective Th1 response against Bordetella pertussis. Infect. Immun. 67 (10), 5372–5378. Coelho, L.H.L., Guimaraes, M.P., Lima, W.S., 2008. Influence of shell size of Lymnaea columella on infectivity and development of Fasciola hepatica. J. Helminthol. 82, 77–80. Cook, D.N., Beck, M.A., Coffmann, T.M., Kirby, S.L., Sheridan, J.F., Pragnell, I.B., Smithies, O., 1999. Requeriment of MIP-1 alpha for an inflammatory response to viral infection. Science 286 (5447), 2098–2102. Daemon, E., Serra-Freire, N.M., 1992. Estudos da relac¸ão custo-benefício em parasitologia: uma proposta de análise. Parasitología al dia 16, 59–62. Dawes, B., 1963a. Hyperplasia of the duct biliar in fasciolosis and its relation to the problem of nutrition in the liver-fluke, Fasciola hepatica L. Parasitology 53, 123–133. Dawes, B., 1963b. The migration of juvenile forms of Fasciola hepatica L through the wall of the intestines in the mouse, with some observations on food and feeding. Parasitology 53, 109–122. Dawes, B., 1963c. Some observations of Fasciola hepatica L. during feeding operations in the hepatic parenchyma of the mouse, with notes on the nature of liver damage in this host. Parasitology 53, 135–143.
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