Changes on Schistosoma mansoni (Digenea: Schistosomatidae) worm load in Nectomys squamipes (Rodentia: Sigmodontinae) concurrently infected with Echinostoma paraensei (Digenea: Echinostomatidae)

Changes on Schistosoma mansoni (Digenea: Schistosomatidae) Worm Load in Nectomys squamipes (Rodentia: Sigmodontinae) Concurrently Infected with Echinostoma paraensei (Digenea: Echinostomatidae)

Vol. 96, Suppl.: 193-198, 2001

Arnaldo Maldonado Júnior/⁺, Renata Coura, Juberlan da Silva Garcia, Reinalda Marisa Lanfredi*, Luis Rey

Laboratório de Biologia e Controle da Esquistossomose, Departamento de Medicina Tropical, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil *Laboratório de Helmintologia, Programa de Biologia Celular e Parasitologia, Instituto de Biofísica Carlos Filho, UFRJ, Rio de Janeiro, RJ, Brasil

The water rat, Nectomys squamipes, closely involved in schistosomiasis transmission in Brazil, has been found naturally infected simultaneously by Schistosoma mansoni and Echinostoma paraensei. Laboratory experiments were conducted to verify parasitic interaction in concurrent infection. It was replicated four times with a total of 42 water rats and essayed two times with 90 mice pre-infected with E. paraensei. Rodents were divided into three groups in each replication. A wild strain recently isolated from Sumidouro, RJ, and a laboratory strain of S. mansoni from Belo Horizonte (BH) was used. Rats infected with E. paraensei were challenged 4 weeks later with S. mansoni and mice 2 or 6 weeks after the infection with S. mansoni. Necropsy took place 8 weeks following S. mansoni infection. The N. squamipes treatment groups challenged with S. mansoni RJ strain showed a significant decrease (80 and 65%) in the S. mansoni parasite load when compared with their respective control groups. There was a significant change or no change in the hosts challenged with the BH strain. The persistence time of E. paraensei within host was extended in relation to control groups, with a consequent enhancement of the number of recovered worm. An E. paraensei strain-specific influence on S. mansoni parasitism is reported. This paper presents some experimental data about this interaction in N. squamipes and Mus musculus.

Key words: Nectomys squamipes - Schistosoma mansoni - Echinostoma paraensei - concurrent infection - heterologous interaction

The co-existence of helminthes in vertebrate hosts has been extensively investigated. Simultaneous infections of two or more helminthes species commonly occur in domestic animals and humans (Christensen et al. 1987). This raises the possibility that one species may influence the transmission patterns or disease features caused by another (Chamome et al. 1990, Chieffi 1992).

The helminth genus Echinostoma (Digenea: Echinostomatidae) has a large geographic distribution due to their ability to parasitize a variety of invertebrate and vertebrate hosts (Yamaguti 1971, Huffman & Fried 1990). Some species develop their larval stages in Biomphalaria glabrata snails, the most important intermediate host of Schistosoma mansoni in Brazil (Loker & Adema 1995).

Some experimental studies describe effects of interactions between Echinostoma revolutum (synonym E. caproni) and S. mansoni in homologous or heterologous infection of definitive hosts. Mice pre-infected by E. revolutum showed decrease in natural resistance, with an increase of the S. mansoni load in experimental conditions (Christensen et al. 1981, 1987).

The parasitism of S. mansoni in a naturally infected population of water rat Nectomys squamipes in an endemic area (Sumidouro, RJ) was studied by D'Andrea et al. (2000). This rodent is closely involved in schistosomiasis transmission since it is a definitive host of S. mansoni (Rey 1993). N. squamipes was also found naturally infected by E. paraensei in Sumidouro (Maldonado Jr. et al. 2001), after which this species has been maintained in laboratory conditions with a sympatric snail as the intermediate host (Maldonado Jr. et al. 2001).

E. paraensei was described by Lie and Basch (1967) from naturally infected B. glabrata from Belo Horizonte, Brazil. However, little is known about the relationship with its natural vertebrate host. In addition, no literature is available about the experimental infection by E. paraensei on his natural rodent host and its concurrent helminth infection.

This paper presents new information of concurrent infections in N. squamipes and Mus musculus by E. paraensei and S. mansoni. Knowledge of this interaction could be useful to schistosomiasis control programs, since the echinostome is able to interfere in the S. mansoni ability to establish the parasitism.

^{ACKOWLEDGMENTS}

Experimental group - Forty-two N. squamipes reared in our laboratory animal house (D'Andrea et al. 1996) representing both sexes and of about three months of age were used. Additionally, we used 90 male albino mice (M. musculus - Swiss Webster), weighing about 25 g , obtained from the Central Animal House of Fiocruz. The experiment was repeated four times with N. squamipes and twice with the mice. Each replicate contained three treatment groups: two control groups (S. mansoni and E. paraensei) and one experimental group (E. paraensei + S. mansoni = concurrent treatment). For N. squamipes, three specimens were used per treatment in the 1st, 2nd and 4threplicates, and five specimens per treatment in the 3rd group. Fifteen mice were used for each treatment group.

Experimental infection - For E. paraensei infection, animals of experimental and control groups were orally infected by a gastric probe with an inoculums of 50 and 25 metacercariae per rat and mice, respectively. The E. paraensei used was isolated from naturally infected N. squamipes from Sumidouro, RJ, and passed through sympatric B. glabrata. The S. mansoni exposure was done four weeks after E. paraensei exposure. Each experimental and control animal was inoculated transcuta-neously through the tail with about 500 (N. squamipes) and 100 (M. musculus) cercariae per host. In the 1st and 2nd replicates with N. squamipes we used a wild strain of S. mansoni from Sumidouro. However, the rats of 3rd and 4th replicates and the mice were infected with a laboratory strain of S. mansoni from Belo Horizonte, MG. This strain has been maintained in the laboratory since 1985. Mice were challenged with S. mansoni on the 2nd and 6th week after the first infection with E. paraensei. The water rats were lethally anesthetized eight weeks after the challenge with S. mansoni, a total of 12 weeks after the exposure to E. paraensei, while mice were necropsied 10 (1st trial) and 14 weeks (2nd trial). S. mansoni adult worms were recovered by perfusion of the hepatic portal system (Pellegrino & Siqueira 1956), followed by collection of worms from the mesenteric veins. As for E. paraensei, the worms were collected in pancreas and small intestine. The small intestine was divided in five equal parts to allow evaluating the parasite's distribution (Kaufman & Fried 1994).

Statistic analysis - The data were analyzed by Mann-Withney test; T test and variance analysis showed arithmetic mean. Values less than 0.05 (P < 0.05) were statically considered significant.

Experiments were performed according to the laws of the Ethical Commission of Animals Use of Fiocruz.

The N. squamipes pre-infected with E. paraensei and later challenged by the S. mansoni wild strain showed a significant reduction in the establishment of S. mansoni worm burdens of 65% in the 1st and 80% in the 2rd replicate (P < 0.05). An enlargement of E. paraensei's persistence time within the host occurred, with a consequent greater worm number of 150% and 300% (P < 0.05) when compared to N. squamipes exposed to E. paraensei only.

The N. squamipes and mice inoculated with the laboratory strain of S. mansoni, showed no significant changes in the S. mansoni parasite load. On the contrary, in the 3rd replicate, N. squamipes showed an increase of 64% of S. mansoni and 9.3% of E. paraensei. No change in the 4th replicate, in relation to the S. mansoni control was observed (Fig. 1).

Figure 1

An enlargement of 28.5% of S. mansoni and 100% of E. paraensei parasite burden was observed in the 1st mice trial and a reduction of 92.3% of E. paraensei was observed in the 2nd trial (Fig. 2).

Figure 2

In N. squamipes, S. mansoni had a balanced sex ratio (1:1) for wild and laboratory strains. In mice, a male tendency was observed with a ratio of 2.1/1.0 and 1.2/1.0 in control and experimental treatments groups of the 1st trail, respectively, and 2.2/1.0 and 1.0/1.0 in each treatment of the 2nd trial.

The displacement of E. paraensei in the small intestine and pancreatic duct was also evaluated (Table). A tendency for parasites to cluster in the duodenum and jejunum in both hosts was noticed. However, worms were more aggregated in N. squamipes. All of them were found in the first part of the small intestine and pancreatic duct. In mice, worms were more dispersed throughout the small intestine and a low percentage was noticed in pancreatic duct (Table).

Table

The rodent N. squamipes showed different susceptibility to S. mansoni strains when pre-infected with E. paraensei. The wild strain, that is naturally sympatric to E. paraensei, proved to be more susceptible to interference by E. paraensei in its life cycle establishment than the laboratory isolate. This can be affirmed from the significant reduction in the sympatric S. mansoni (RJ strain) adult worms load recovered from N. squamipes pre-infected by E. paraensei. On the other hand, parasitism intensity was not affected when a laboratory isolate of S. mansoni was the challenger.

The rodent N. squamipes has already been tested for resistance development to the BH laboratory strain of S. mansoni. This natural vertebrate host, pre-infected with S. mansoni, facilitated the establishment of the homologous infection with a consequent increase in worm load when re-infection occurred in the acute phase of the first infection, but it is not observed if the challenge happens in the chronic phase (Maldonado Jr. et al. 1994).

A different pattern of susceptibility to S. mansoni infection was noticed within mice trails and between host species, also infected by the laboratory strain. Increase and reduction of S. mansoni parasite load occurred in mice, depending on the time of challenge, 2nd or 6th week after the first infection. However, this oscillatory behavior does not present significant levels. Christensen et al. (1981) and Sirag et al. (1980) have also noticed antagonistic data on S. mansoni recovery, with no parasite load change in a primary, early and low-level E. revolutum (synonim: E. caproni) mice infection or with a significant increase in a primary, late and heavy infection.

Both strains of S. mansoni have no major differences in adult worms proteins in SDS-poliacrilamide gel electrophoresis profiles (Maldonado Jr. & DeSimone 1991). Therefore, a heterogeneous effect of E. paraensei infection to S. mansoni susceptibility could be expected as a consequence of the major polymorphism of the wild strain (Simpson 1995) or by diversity among adult schistosomes, including near as well as far species, confirmed through mitochondrial DNA sequence (Le 2000). The wild strain can be upset by environment pressure because of its genetic heterogeneity. In addition, this strain comes from an endemic area where the parasite circulates within human and wild rodents (D'Andrea et al. 2000) and it presents a heterogeneous phenotype in adult worms and cercariae when isolated from the distinct vertebrate hosts (Neves 1998). Experimentally infected sympatric B. glabrata presented chronobiological behavior with an early and a later cercarial emergence pattern (Machado-Silva 1981). The laboratory S. mansoni strain has been maintained in laboratorial conditions for a long time. Thus, it probably has reduced genetic heterogeneity as that observed in other laboratory maintained parasites (Voltz et al. 1987).

Data showed that E. paraensei is able to influence on S. mansoni parasitism behavior, although it is a strain-specific action. This fact can be explained by many hypotheses. When compared to echinostomatid, S. mansoni has a recent geographic establishment (Rey 1993) and is probably, still adaptting to parasitism in this rodent. Nevertheless, these trematodes are able to co-exist in the same host. These conditions would favour interference by E. paraensei, however, these trematodes set themselves in different niches within the host and their interaction mechanisms can be indirect, by induction of immunological factors as shown in E. caproni infection (Brunet et al. 1999).

Some studies showed a male bias in the S. mansoni sex ratio, in both experimental (Liberatos 1987) and natural infections (Barral et al. 1996). This can be associated to a potential male cercariae infection greater than that of female cercariae (Bossier et al. 1999). In agreement with these finding, our data shows a male bias in mice. This is a phenomenon independent of infection time, yet influenced by concurrent infection. The initial infection by E. paraensei seems to favor males more than female.

Usually, S. mansoni infections are permanent while E. paraensei infections lasts at least 12 weeks in N. squamipes and worms are gradually eliminated during this time. The increased persistence of the worm load of E. paraensei within hosts harboring S. mansoni suggests that the adult schistosome infections are probably down-regulating the immune response of the host (Fallon & Dunne 1999). Fujino et al. (1993) suggested a non-immunological mechanism of E. caproni and E. trivolvis expulsion that are dependent of mucin secretion increase by intestinal goblet cells. Although it was not possible to establish the exact modulator mechanism yet, data suggested that S. mansoni probably delay the mechanisms of E. paraensei expulsion, which can be mediated immunologically, either directly or indirectly (Christensen 1981), favouring the echinostomatid survival.

Echinostomatids have specific niches within the small intestine of their definitive hosts. They are typically dispersed in the initial phases of infection (Nollen 1996). E. paraensei commonly occurs in the duodenum-jejunal. From our data, the distribution does not change when the host is challenged by S. mansoni. However, host species can influence on E. paraensei distribution, as shown by the predilection of E. paraensei to occupy the pancreatic duct of N. squamipes when compared to M. musculus.

There are evidences that both adult worms and larvae stages of E. paraensei (in B. glabrata) can potentially interfere on the survival of S. mansoni. Some studies have proposed the use of larvae of E. paraensei is schistosomiasis biological control, since Echinostoma spp. rediae attack and destroy S. mansoni sporocists by cannibalism or induction of host's inflammatory response, when they are in the same molluscan (Lie 1973, Jourdane et al. 1990, Adema et al. 1999).

We conclude that the presence of E. paraensei interferes with the development of a wild strain of S. mansoni (RJ strain), changing its relationship with the host. However, the bases of the interaction mechanism are still being evaluated to be considered in schistosomiasis control programs.

To Dr Eric S Loker for his valuable comments on the manuscript.

Fig. 1 | Fig. 2 | Table

This work was supported in part by Fundação Oswaldo Cruz-Papes, Faperj, CNPq, FUJB and Pronex.

References

• Adema CM, Hertel LA, Loker ES 1999. Evidence from two planorbid snails of a complex and dedicated response to digenean (echinostome) infection. Parasitology 119: 395-404.
• Barral V, Morand S, Pointier JP, Théron A 1996. Distribution of schistosome genetic diversity within naturally infected Rattus rattus detected by RAPD markers. Parasitology 113: 511-517.
• Boissier J, Morand S, Moné H 1999. A review of performance and pathogenicity of male and female Schistosoma mansoni during the life cycle. Parasitology 119: 447-454.
• Brunet LR, Joseph S, Dunne DW, Fried B 2000. Immune responses during the acute stages of infection with the intestinal trematode Echinostoma caproni. Parasitology 120: 565-571.
• Chamome M, Marques CA, Atuncar GS, Pereira ALA, Pereira LH 1990. Are there interactions between schistosomes and intestinal nematodes? Trans R Soc Trop Med Hyg 84: 557-558.
• Chieffi PP 1992. Interrelashionship between schistosomiasis and concomitant diseases. Mem Inst Oswaldo Cruz 87 (Suppl. IV): 291-296.
• Christensen NO, Nansen P, Fagbeni BO, Morand J 1987. Heterologous antagonistic and synergistic interactions between helminths and between helminths and protozous in concurrent experimental infection of mammalian hosts. Parasitol Res 73: 387-410.
• Christensen NO, Nydal R, Frandsen F, Nansen P 1981. Homologous immunotolerance and decreased resistance to Schistossoma mansoni in Echinostoma revolutum infected mice. J Parasitol 67: 164-166.
• D' Andrea PS, Horta C, Cerqueira R, Rey L 1996. Breeding of the water rat (Nectomys squamipes) in the laboratory. Lab Animal 30: 369-376.
• D' Andrea PS, Moroja LS, Gentile R, Cerqueira R, Maldonado Jr. A, Rey L 2000. The parasitism of Schistosoma mansoni (Digenea-Trematoda) in a naturally infected population of water rat, Nectomys squamipes (Rodentia: Sigmondontinae) in Brazil. Parasitology 120: 573-582.
• Fallon PG, Dunne DW 1999. Tolerization of mice to Schistosoma mansoni egg antigens causes elevated type1 and diminished type 2 cytokine responses and increased mortality in acute infection. J Immunol 162: 4122-4132.
• Fujino T, Fried B 1993. Expulsion of Echinostoma trivolvis (Cort, 1914) Kanev, 1985 and retention of Echinostoma caproni Richard, 1964 (Trematoda: Echinostomatidae) in C3H mice: pathological ultrastructural and cytochemical effects on the host intestine. Parasitol Res 79: 286-292.
• Huffman JE, Fried B 1990. Echinostoma and echinostomiasis. Adv Parasitol 29: 215-269.
• Jourdane J, Mounkassa JB, Imbert-Establet D 1990. Influence of intramolluscan larval stages of Echinostoma liei on the infectivity of Schistosoma mansoni cercariae. J Helmintol 64: 71-74.
• Kaufman AR, Fried B 1994. Infectivity, growth, distribution and fecundity of a six versus twenty-five metacercarial cyst inoculum of Echinostoma caproni in ICR mice. J Helmintol 68: 203-206.
• Le TH, Blair D, McManus DP 2000. Mitochondrial DNA sequences of human schistosomes: the current status. Inter J Parasitol 30: 283-290.
• Liberatos JD 1987. Schistosoma mansoni: malebiased sex ratios in snails and mice. Exp Parasitol 64: 165-177.
• Lie KJ 1973. Larval trematode antagonism: principles and possible application as a control method. Exp Parasitol 33: 343-349.
• Lie KJ, Basch PF 1967. The life history of Echinostoma paraensei sp. n. (Trematoda: Echinostomatidae). J Parasitol 53: 1192-1199.
• Loker ES, Adema CM 1995. Schistosomes, echinostomes and snails: comparative immunobiology. Parasitol Today 11: 120-124.
• Machado-Silva JR 1981. Observaçőes sobre a Evoluçăo de Diferentes Amostras de Schistosoma mansoni em Biomphalaria glabrata e Mus musculus, Master Sci Thesis, Instituto Oswaldo Cruz, Rio de Janeiro, 103 pp.
• Maldonado Jr. A, Simone SG 1991. Identification and characterization of sex-linked proteins of Schistosoma mansoni. Mem Inst Oswaldo Cruz 86: 31-36.
• Maldonado Jr. A, Locker ES, Morgan JAT, Rey L, Lanfredi RM 2001. Description of a new Brazilian isolate of Echinostoma paraensei (Platyhelminthes: Digenea) from its natural vertebrate host Nectomys squamipes by light and scanning electron microscopy and molecular analysis. Parasitol Res (in press).
• Maldonado Jr. A, Machado e Silva JR, Rodrigues e Silva R, Lenzi HL, Rey L 1994. Evaluation of the resistance to Schistosoma mansoni infetion in Nectomys squamipes (Rodentia: Cricetidae), a natural host of infetion in Brazil. Rev Inst Med Trop Săo Paulo 36: 193-198.
• Maldonado Jr. A, Vieira GO, Garcia JS, Rey L, Lanfredi RM 2001. Biological aspects of a new isolate of Echinostoma paraensei (Digenea: Echinos-stomatidae): susceptibility of sympatric snails and the natural vertebrate host. Parasitol Res (in press).
• Neves RH, Pereira MJS, Oliveira, RMF, Gomes DC, Machado-Silva JR 1998. Schistosoma mansoni Sambon, 1907: morphometric differences between adult worms from sympatric rodent and human isolates. Mem Inst Oswaldo Cruz 93: 309-312.
• Nollen PM 1996. Mating behavior of Echinostoma caproni and E. trivolvis in concurrent infections in hamsters. Int J Parasitol 27: 71-75.
• Pellegrino J, Siqueira AF 1956. Técnica de perfusăo para coleta de Schistosoma mansoni em cobaias experimentalmente infectadas. Rev Bras Doenças Trop 8: 589-597.
• Rey L 1993. Non-human vertebrate host of Schistosoma mansoni and schistosomiasis transmission in Brazil. Res Rev Parasitol 53:13-25.
• Simpson AJG 1995. DNA polymorphism of schistosomes and their snail hosts. Mem Inst Oswaldo Cruz 90: 211-213.
• Sirag SB, Christensen NO, Frandsen F, Monrad J, Nansen P 1980. Homologous and heterologous resistence in Echinostoma revolutum infections in mice. Parasitology 80: 479-486.
• Voltz A, Richard J, Pesson B 1987. A genetic comparison between natural and laboratory strains of Echinostoma (Trematoda) by isoenzymatic analysis. Parasitology 95: 471- 477.
• Yamaguti S 1971. Synopsis of Digenetic Trematodes of Vertebrates, Keigaku Publishing Co., Tokyo, 1074 pp.

Publication Dates

• Publication in this collection
  03 Oct 2001
• Date of issue
  Sept 2001

History

• Received
  14 May 2001
• Accepted
  25 July 2001