Juliana de Fátima Macedo SantosI; Joelma VasconcelosI; Joelma Rodrigues de SouzaII; Eridan de Medeiros CoutinhoII; Silvia Maria Lucena MontenegroII; Eulália Azevedo-XimenesI
IDepartment of Antibiotics of the Federal University of Pernambuco, Recife, PE, Brazil IIDepartment of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, PE, Brazil
ABSTRACT
C57Bl/10 male mice infected with Schistosoma mansoni were distributed into mixed, prophylactic and curative groups. A culture of Zymomonas mobilis was orally administered to mice. A 61% protection from the infection was observed in the curative group (p <0.05). Histopathological study of the livers and intestines showed similar results.
Key-words: Zymomonas mobilis. Schistosoma mansoni. Immunopotentiators. Experimental studies.
RESUMO
Camundongos C57Bl/10 do sexo masculino, infectados com Schistosoma mansoni foram distribuídos nos grupos misto, profilático e curativo. Cultura de Zymomonas mobilis foi administrada oralmente aos camundongos. Uma proteção de 61% foi observada no grupo curativo (p<0,05). Os estudos histopatológicos dos fígados e intestinos mostraram resultados similares.
Palavras-chaves: Zymomonas mobilis. Schistosoma mansoni. Imunopotenciadores. Estudos experimentais.
The immune response can be non-specifically stimulated by a number of agents9 12 . One of these, Zymomonas mobilis, has already been studied6, and its antagonistic effects against bacteria, fungi and protozoa are known6 7.
Although Z. mobilis has been used in several biological systems7, little is known about its action on helminthic infections. Schistosomiasis was studied on account of the severity the disease assumes in many cases and the organic deficiency it produces, making it one of the most serious public health problems.
The present study focused on the evaluation of host protection and the granulomatous lesions around Schistosoma mansoni eggs.
Six to eight week old C57Bl/10 male mice were obtained from the animal facilities of CPqAM-Fiocruz, Recife, PE, Brazil. Experimental and control groups were infected percutaneously with 100 cercariae (BH strain) shed from laboratory infected Biomphalaria glabrata.
A standard 109 CFU/mL culture of Zymomonas mobilis was administered orally, at a dose of 0.3mL/day 8.
For evaluation of the resistance to the S. mansoni infection and granuloma formation, the experimental groups were divided into prophylactic (n = 18) and curative (n = 17) in accordance with the guidelines for the administration of the Z. mobilis culture, for 7 days before or 7 days after the infection, respectively. A mixed group (n = 15), receiving the bacterial culture before and after S. mansoni infection was also studied.
In control 1 group (n = 17), mice were infected in a similar way to the experimental group2 but did not ingest Z. mobilis culture. In control 2 group (n = 6), mice were free of infection, but ingested the bacterial culture. In the placebo group (n = 5), the animals ingested culture medium without bacteria and acted as controls for the control 2 group.
The degree of protection was measured by infecting the mice and perfusion of the liver 60 days later. The number of worms recovered from each treated group of mice was compared with the non-treated control group, and protection was expressed as a percentage according to the following formula:
Protection = C-E/C x 100, where:
C – is the number of worms in the control group;
E – is the number of worms in the treated group.
Mice from the experimental and control 1 groups were sacrificed on the 60th day of infection and samples from the liver and intestines were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5µm and stained with hematoxylin-eosin.
Statistical comparisons were made using Student’s t test, with a level of significance for the p value set at 0.05. All the experiments were repeated three times.
The total number of adult worms recovered was significantly lower in the curative group as compared to the control 1 group (p = 0.00049), resulting in 61% protection (Table 1). In this group the immune response elicited by Z. mobilis occurred after the S. mansoni infection. As the activation of the immune system begins on the third day, tends to peak on the fifth and decrease on the eighth day11, the immune response was very efficient, with death of the parasites occurring at the peak phase. The degree of protection reported is comparable to the immunity conferred by some schistosomiasis vaccine candidates3. Previous studies carried out with the immunopotentiators Corynebacterium parvum1 13 and levamisole10 using a similar methodology, demonstrated that the effects on the curative group treated with levamisole were similar to those obtained with the culture of Z. mobilis and different from the results with the use of C. parvum. The variation in the effects of immunopotentiators depend on several factors such as: host immune status, severity of infection, dose, timing of drug administration and specific action of each one1 13.
Therefore, the prophylactic group was not significantly different when compared to the control 1 group, showing 24% protection against S. mansoni infection. In this group, which was treated before the infection, the immune response seemed to be much less efficient. On the other hand, in the mixed group a decrease in the number of adult worms was to be expected, since the onset of the curative treatment would provoke a secondary immune response. However, in this group, which involved a combination of the two treatments (prophylactic and curative), an exacerbation of the schistosomiasis infection was observed, since a larger number of adult worms was collected in comparison with the control 1 (infected and non-treated) group.
Studies with probiotic bacteria demonstrated that the dose and frequency of administration of these preparations influence the course of the stimulation of the immune system5. In this study, Z. mobilis culture dose administrated daily to the mixed group was similar to that given to the prophylactic and curative groups, but there was an increase in the frequency of administration, from 7 to 14 days. The observed increase in frequency may have led to an anergy state in this group, allowing a greater number of adult worms to be recovered at the time of the perfusion.
As far as histopathology was concerned, the lesions were similar in the experimental and control 1 groups. Hepatic changes were characterized by numerous periovular granulomas, mainly of the exudative type. Eggs and granulomas were seen in all the histological layers, in the three intestinal segments, though were more abundant in the walls of the jejunum-ileum.
No statistically significant difference was observed in the weights of the spleen and liver of the mice from the groups infected with S. mansoni. The same was true for the groups that were not infected (Table 2). The latter results in combination with the histopathological analysis of these organs in the control 2 (treated and non-infected) and placebo (non-treated and non-infected) groups, corroborate the conclusion reached by Fox4 that probiotics administered orally do not pose a threat of damage to the liver and spleen.
ACKNOWLEDGEMENTS
We are grateful to Luiz Carlos Figueiredo and Roni Evêncio for technical support.
REFERENCES
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Correspondence to
Drª Silvia Montenegro
Department of Immunology, Centro de Pesquisas Aggeu Magalhães/CPqAM/Fundação Oswaldo Cruz
Av. Moraes Rego s/n
Cidade Universitária, 50670-420 Recife, PE, Brazil
Fax: 55 81 3453-1911 – Tel: 55 81 2101-2565
e-mail: silvia@cpqam.fiocruz.br
Recebido para publicação em 26/2/2004
Aceito em 25/8/2004
Ethics Committee in Use of Animal (CEUA/Fiocruz) n0: P0091-01
This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil).
