IMMUNE RESPONSE OF STIMULATED BIOMPHALARIA ALEXANDRINA SNAILS WITH SCHISTOSOMA MANSONI INFECTION.

In this study, both digestive and hermaphrodite glands in Biomphalaria alexandrina snails were examined by light microscope (LM) and scanning electron microscopy (SEM) in a trial to clarify its immunological role as defense mechanism against the parasite infection. B. alexandrina snails used were exposed individually to Schistosoma mansoni miracidia; according to their responsq they were classified into infected snails (shed cercariae) and non-infected snails (failed to shed cercariae). Snails not exposed to S. mansoni miracidia used as control. LM showing great histological alteration in tissues, of the both glands in infected snails, these changes represented by degenerated both oocyte and spermatocyte in hermaphrodite gland in addition to degenerated digestive gland while non-infected snails showing mild degenerated ova with normal spermatocytes also degenerated miracidia. inside granuloma like structure with concentric layers of fibroblast and haemocyte could be observed. By SEM we could detect extensively damaged and fibrosed non-motile cilia with exfoliation of tegumental surface in in- fected B.alexandrina while non-infected ones showing attached numerous cilia with some ballooning of the folds.

Ultrastructural study on Biomphalaria alexandrina haemocytes infected with Schistosoma mansoni in Egypt and its correlation with nitric oxide level.

Some snails of Biomphalaria alexandrina can resist the infection of Schistosoma mansoni so this study aimed to clearly this mechanism by using light and electron microscopy (EM) and determine the role of Nitric oxide in this mechanism. B. alexandrina snails used in this study were exposed individually to S. mansoni infection according to their response they were classified into susceptible group (shed cercariae) and resistant group (failed to shed cercariae). Snails not exposed to infection were included in this study as control group. Nitric oxide (NO) level was assayed directly in the soluble fraction of B. alexandrina haemolymph supernatants collected from each group of B. alexandrina snails were subjected to NO assay by the Greiss reaction. The level of NO in haemolymph of infected snails was significantly increased (p < 0.001) than both control and non infected snails groups, however, in non infected snails group had significantly (p < 0.05) compared to control group. This study when correlated the changes recognized by EM with NO level the pro apoptotic effect of high level of NO on the haemocytes. Characterization and identification of cell shape of haemocytes in both haemolymph and tissue were examined by light and electron microscopy. Examination of B. alexandrina snail's haemocytes revealed three types of different cells classified according to their shape and granular contents. These cells are granulocytes, amoebocytes and hyalineocytes. Electron microscope study also revealed the important role of granulocytes and amoebocytes as defense mechanism against snail infection. NO is considered an important anti parasite molecule; intra-molluscan stages of parasites switch off host NO defense response.

Susceptibility of Biomphalaria spp. to infection with Schistosoma mansoni in sympatric and allopatric combinations with observations on the genetic variability between snails.

This investigation was carried out to study the susceptibility of Saudi Biomphalaria arabica to Egyptian Schistosoma mansoni in comparison with the susceptibility of Egyptian Biomphalaria alexandrina to the same parasite. This was in order to know the possibility that the parasite might be able to spread into Saudi Arabia and to determine the genetic variability between Egyptian B. alexandrina and Saudi Biomphalaria arabica snails. Lab bred Egyptian B. alexandrina and Saudi B. arabica snails were exposed individually to 10 freshly hatched Egyptian S. mansoni miracidia/snail. The mortality rate, infection rate, prepatent period, duration of cercarial shedding and cercariae production per snail were recorded in both the sympatric couple (Egyptian B. alexandrina and Egyptian S. mansoni) and in the allopatric combination (Saudi B. arabica and Egyptian S. mansoni). The results revealed that, the survival rate of snails exposed to Egyptian S. mansoni miracidia at 34th day post-exposure (at first cercarial shedding) was higher in B. arabica than in B. alexandrina. After shedding, the mortality rate was higher in the B. arabica, compared to B. alexandrina. The infection rate was higher in B. arabica than B. alexandrina; the mean of prepatent period was shorter in the B. arabica than in the B. alexandrina. However, the duration of cercarial shedding was longer in the Egyptian snails and the cercarial production per snail was higher in B. alexandrina snails than in B. arabica. To study the genetic variability between B. alexandrina and B. arabica, RAPD-PCR on the genomic DNA of snails was done. RAPD-PCR revealed significant variation between the two snail species. In conclusion, the results suggest that B. arabica can play a role in the transmission of Egyptian S. mansoni in Saudi Arabia and therefore this parasite might be able to spread into the Kingdom. In addition, the RAPD-PCR results demonstrated genetic variability between the two species which may be related to the differences in susceptibility of both Saudi and Egyptian Biomphalaria snails to Egyptian S. mansoni infection.

Immunolocalization of Schistosoma mansoni and Schistosoma haematobium antigens reacting with their Egyptian snail vectors.

The reaction of the haemolymph and the tissue of infected intermediate hosts, Biomphalaria alexandrina and Bulinus truncatus to Schistosoma mansoni and S. haematobium antigens were investigated using the indirect immunoperoxidase technique. A new technique, Agarose cell block was used in collection of haemolymph which helped in collecting plenty of well formed cells in comparison to the ordinary one using the cytospin. Collected haemolymph and prepared tissues of uninfected and infected B. alexandria and B. truncatus were fixed and then reacted with anti-S. mansoni and anti-S. haematobium IgG polyclonal antibodies. The haemolymph and tissue of infected B. alexandrina and B. truncatus gave a positive peroxidase reaction represented by a brown colour. In haemolymph, the positive peroxidase reaction was detected mainly in the cytoplasm of the amoebocytes. In the tissue, it was detected in epithelial cells lining the tubules, male cells in the lumen of the tubules and in female oogonia cells along the periphery of the tubules. The similarity in the strength and distribution of positive reaction in B. alexandrina and B. truncates was observed as compared to control. Thus, the immunoperoxidase technique proved to be an effective indicator for the schistosome-antigen in the snails.

Electrophoretic patterns of protein fractionations in hemolymph and tissues of Biomphalaria alexandrina and Bulinus truncatus during course of schistosome infection.

Electrophoresis of plasma protein of B. alexandrina (uninfected & infected with S. mansoni) showed that the major dominant bands had molecular weights of 20, 44, 96, 139 & 205 KD in both types of snails. The 1day & 1 week post miracidial exposure (PME) groups were characterized by band 54 KD. All groups except a day PME were characterized by a common band of MW 65 KD. Three days PME group had. three bands of 123 KD, 150 &177 KD, not found in other groups. The highest similarity index in 2 weeks PME & 5 weeks PME groups (during cercarial shedding) was 0.667 and the lowest one was in 3-days PME (0.5). The 3-days PME had a unique band of MW 177.04 KD, not found in other groups. Similar electrophoretic pattern of B. alexandrina tissue protein was seen. The major dominant bands had molecular weights of 14, 21, 80 and 140 KD in both non-infected and infected snails. The 1day PME had a band of 48.483 KD, 3-days PME had a band of 87.985 KD, one-week PME group characterized by two bands 61.761 KD and 70.338 KD. The two-weeks PME had a band 91.111 KD. While, the 5 week PME (during cercarial production) was the only group that shared the common band of MW 115 KD with controls. The highest similarity index in 5 weeks PME (during cercarial shedding) group was 0.545 and the lowest one was in 1 week & 2 weeks PME (0.43). The electrophoresis of plasma protein of B. truncatus (uninfected & infected with S. haematobium) showed that the major dominant bands had molecular weights of 20, 30, 65, 80, 106, 117 & 170 KD in both type of snails. The 1day PME group was characterized by three bands of MWs 26.539, 51.891 & 91.509 KD. All experimental groups, except 5 weeks PME (during cercarial shedding) and control, had a common band of MW 45 KD. Three days PME group had a characteristic band of 113.72 KD which was not found in any other group. The highest similarity index was in one week PME group was 0.857 and the lowest one in 1-day PME (0.5). In B. truncatus tissue protein, the major dominant bands by electrophoretic pattern had molecular weights of 20, 45, 54, 80, 97 & 171 KD in both type of snails. A day PME had a band of 73.544 KD and a week PME had a band of MW 60.813 KD. Two and 5 weeks PME groups had 2 bands of MWs 27 & 62 KD. All experimental groups had a characteristic band not found in control of MW 141 KD. The highest similarity index in 3-days PME was 0.8 and the lowest one was in 5 weeks PME during cercarial shedding(0.545).

Morphological and ecological studies on Lymnaea natalensis the snail vector of Fasciola gigantica in Egypt.

Lymnaea natalensis were collected from several localities in Giza Governorate (El-Mansouria, Kafr Hakim and The Nile). The collected snails were examined for cercarial shedding and measurement of shell was carried out using a virner caliper. A total of 217 Lymnaea was collected from all habitats and 24 snails were found to shed Fasciola gigantica cercariae with infection rate 11.1%. The ratio of shell height to shell width (H/W) ranged 1.7-2.1. The ratio between length of aperture to shell height (A/H) was almost constant ranged 0.7-0.8. While the ratio between length of aperture and shell width (A/W) ranged from to 1.8. There was a significant positive correlation between shell height and both shell width and length of aperture (p<0.001). The same relation was clearly indicated between shell width and length of aperture. Also, when the aperture length increased the ratio between height and width increased (p<0.05). While, the following ratios (H/W), (A/H) and (A/W) remained somewhat constant irrespective to the number of whorls. There was a positive correlation between number of whorls and each of aperture length, shell length and shell height. On maintaining L. natalensis in different pH values, the results showed that at pH 3 and pH 11, all snails maintained at these pH values died after one day of maintenance. While snails survived and laid eggs in the range of pH 5-9.