This study aims to evaluate the susceptibility of the hedgehog (Hemiechinus auritus) to Schistosoma mansoni under the experimental condition. The susceptibility of this animal to S. mansoni infection was compared to the hamster (Permissive host) infection with the same parasite. In this study, 24 male of both hedgehogs and hamsters were used. Half of animals were infected with 250 S. mansoni cercariae. Eight weeks post infection; animals were sacrificed to determine the total worm burden, total eggs count, granuloma volume. The results revealed that a fecal examination of the infected hedgehogs showed no eggs were detected in the feces. The average of hamsters pre-patent period (the onset of patency) was 44.0±1.0 days. The average of S. mansoni worms recovered from hedgehogs was 31.0±10.0 worms, while the worm recovered from hamsters was 82.0±18.0. The granuloma volume measured from hedgehogs and the hamsters were 74.0±56.0 and 119.0±75.0, respectively. Marked reduction in the some biochemical parameters after the infection with S. mansoni was recorded in infected hedgehogs and hamsters. In conclusion, the hedgehogs showed non possibility for this animal to be a risk for schistosomiasis in the field.
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Schistosomiasis is an endemic disease in tropical and subtropical areas representing the second in importance to malaria from the socioeconomic point of view. It is endemic in 74 developing countries and effected between 391 to 597 million people worldwide (King, 2010). Recent studies showed that over 800 million, mostly children are at risk of infection (Gray et al., 2010; Siddiqui et al., 2011; Omonijo et al., 2013). In particular, the risk of infection in Egypt is still high due to the presence of S. haematobium and S. mansoni in the endemic areas of delta (El-Khoby et al., 2000), middle and Upper Egypt (Hammam et al., 2000). In Egypt, the epidemiology of schistosomiasis has been extensively investigated (Hotez, 2009), since the distribution and prevalence were documented for most affected areas (Gabr et al., 2000; Hammam et al., 2000). In recent study, over 89,000 persons in 251 rural communities found to be infected. In 4 governorates, the prevalence of S. mansoni where it is endemic averaged 36.4% while S. haematobium where it is endemic average 7.8 % [http://www.pharmawriterpro. com/high-Prevalence-of schistosomiasis-in-Egypt.html].
So far, control of schistosomiasis depends mainly on praziquantel (PZQ) which becomes the drug of choice for the treatment worldwide. Due to the extensive uses of this drug as the only drug of choice, raise a question about the possibility of its resistance development, therefore, researchers have been trying to break down this resistance (Lar and Oyerinde, 2007). Furthermore, controversy studies showed that the new anti-schistosomal drug Mirazid may or may not be suitable to replace PZQ (Abdel-Aziz et al., 2006; El-Kott et al., 2011). Finding a potential vaccine for schistosomiasis is still under trials (Bashtar et al., 2006; Maghraby et al., 2007; Soliman, 2008).
It is well known that human is the most definitive host of S. mansoni and S. haematobium for maintenance of the life cycle, however, several studies showed that other mammalian species may contribute in maintaining the life cycle as well (Costa-Silva et al., 2002; El-Naggar et al., 2011). Several studies have been reported that most of the mammals with natural infection with S. mansoni were rodents, chimpanzee (Gentile et al., 2006; El-Naggar et al., 2011). In Egypt foci, however, it has found that the gerbil, the Nile rats and the field rats were naturally infected with both of S. mansoni and S. haematobium (Mansour, 1973). Recently, it has reported that under the experimental conditions, wild rodent species including Nile rats, house rats, Cairo spiny mice, black mice (Sisi) and field rats were found to be infected with S. mansoni and could be able to close the life cycle similar to human (El-Naggar et al., 2011). Similarly, in Brazil, it has found that the water-rats; Nectomys squamipes may present different levels of importance in the transmission of S. mansoni and could be considered important wild-reservoirs to this disease (DAndrea et al., 2002; Gentile et al., 2006).
Hemiechinus auritus (the hedgehog) is a common insectivore in Egypt and it is widely distributed all over the Nile Delta. Its susceptibility to S. mansoni infection was not investigated yet. The present study was conducted to primarily evaluate its sensitivity to S. mansoni infection and to what extend the hepato-pathological changes may affects the life span of the affected animals.
MATERIALS AND METHODS
Hedgehogs (Hemiechinus auritus) and hamsters: Hedgehogs, Hemiechinus auritus, belong to order Insectivora, family Erinaceidae (Strocker, 2005). Twenty four male hedgehogs of body weight of 202.0±29.0 g were collected from the field and used in this study. Animals were treated with a single dose of praziquantel (PZQ), 2 weeks before the experimentation; therefore these animals were free from helminthic infections as judged by fecal examinations. They were acclimatized to the laboratory conditions for 2 weeks before experimentation and they maintained on laboratory standard balanced diet. Twelve males of the laboratory permissive host Syrian golden hamsters, Mesocresitus auratus were used as a control group. Hamsters of body weight of 92.0±13.0 g were obtained from the Experimental Animal Research Unit of the Schistosome biological supply program at Theodor Bilharz Research Institute, Giza, Egypt. Hedgehogs and hamsters were infected and housed under the laboratory condition at the animal facility of the Department of Zoology, Faculty of Science, Tanta University, Egypt.
Parasite and animals infection: Schistasoma mansoni cercariae maintained in Biomphalaria alexandrina were used in this study. The parasite life cycle was kept in the laboratory according to the method described by Christensen et al. (1984). Twelve hedgehogs and twelve hamsters were individually exposed to 250 S. mansoni cercariae for 2 h, by partial immersion of their body into the cercarial suspension (Christensen et al., 1984). Twelve animals of each species were saved as normal controls.
In a different experiment, hamsters and hedgehogs were used to determine the survival rate upon 9 weeks post-infection with 250 S. mansoni cercariae.
Determination of the pre-patent period and the fecundity of female worms: Coprologic study was conducted to determine the pre-patent period of S. mansoni in the infected animals (the time from initial infection to the first appearance of eggs in the animal feces), in addition to the fecundity of female worms (the mean number of eggs produced per female worm per day). The pre-patent period was determined by daily inspection of the feces, according to the technique of Marshall et al. (1989), started from the 35th day post-infection. The fecundity of S. mansoni female was obtained by determination of the number of eggs per gram of feces every 3 days over a period of 2 weeks started from the 38th day post-infection. The fecundity was determined by dividing the average daily egg output in the feces of the animals by the number of mature female worms recovered.
Enumeration of males, females and the total worm burden: Eight weeks post infection, the animals were sacrificed. Briefly, to sacrifice hedgehogs, individual animals were anesthetized with suitable anesthesia to relax the abdominal muscle. Each animal was sacrificed and livers were perfused according to the technique of Christensen et al. (1984). Using normal saline containing anti-coagulant, liver of each animal was perfused to eliminate single and coupled worms from the liver. All worms were picked and transferred into clean petri dishes to be counted under dissecting microscopes. The mesenteric veins were then examined for picking any remaining adult worms trapped in.
Enumeration of eggs lodged in different organs: Tissue samples, each of 0.1 g, were excised from the lung, spleen, liver and intestine of the infected animals and then digested in 5% KOH solution. The number of eggs g-1 of tissue was estimated.
Granuloma measurements: For histo-pathological examination, selected liver specimens were fixed in 10% neutral formalin paraffin, sections of 6 μm thickness were prepared and then stained with hematoxylin and eosin methods. Lesions containing single eggs in their centers were selected for measurement and the diameter of each granuloma was obtained by measuring two diameters of the lesion at right angles to each other using an ocular micrometer. The mean diameter of at least 70 lesions from the hedgehog of each species was determined and the volume of each lesion was calculated, assuming a spherical shape (Cheever et al., 1993), from its mean diameter using the following equation:
Determination of the biochemical assays: Determination of the liver cholinesterase activity was estimated according to Ellman et al. (1961), arginase (Brown and Cohen, 1959). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed according to Reitman and Frankel (1957). In addition, total lipids (Frings et al., 1972), total protein (Lowry et al., 1951) in the liver homogenate and serum albumin (Doumas et al., 1971) were determined.
Statistical analysis of the data: Students t- test was used to compare the mean volumes of the granulomas and the biochemical parameters measured. Values of p<0.05 were considered statistically significant.
Survival rate of infected animals: Exposure of hedgehogs and hamsters individually to S. mansoni infection at a dose of 250 cercariae per animal resulted into marked mortalities. Hedgehog was much more affected than hamster where 10 hedgehogs out of 24 exposed to the infection survived by the end of the observation period (9 weeks) whereas the number of hamster survived were 12 out of the 15 exposed to the infection (Fig. 1).
The onset of patency of S. mansoni infection and the fecundity of female worms: Fecal examinations of hedgehogs exposed to 250 S. mansoni cercariae per animal showed that no eggs were detected in the feces of the hedgehogs whereas the average pre-patent period (the onset of patency) was 44±1 days for the hamster. No significant difference was observed among the 5 fecal examinations carried out for the hamster (H = 0.7, p = 0.9) (Table 1).
|Table 1:||No. of eggs per gram of feces (epg) of the hamsters and hedgehogs in five coprologic examinations|
|SmF: S. mansoni female, SD: Standard deviation|
|Fig. 1:||Survival curve of S. mansoni infected hamsters and hedgehogs. Hamsters and hedgehogs were infected with 250 cercaria/animal and were housed for 9 weeks under experimental conditions|
Also, the number of eggs per gram of feces did not correlate significantly with the number of female worms recovered (rs = 0.02, p>0.05). Miracidial hatching tests showed that the eggs recovered from tissue of the hedgehog were viable and infective. The same observation was recorded for the hamster.
The hatching meracidia were used to infect clean snails (Bimophalaria alexandrina) and interestingly, snails infected with meracidia emerged from S. mansoni eggs harvested from either hedgehogs or hamster produced infective cercariae which were able to infect albino mice again.
The total worm burden recovery after the infection with S. mansoni cercariae: To address the capability and the susceptibility of hedgehogs to S. mansoni infection, after 8 weeks post-infection, all exposed animals to S. mansoni were sacrificed and the numbers of male, female and total worm number were assessed through liver perfusion and picking of any remaining worms from the intestinal mesenteries. The results showed that significant differences in the number of adult worms recovered between the two species were found. The hedgehogs were found to be loaded with 31.0±10.0 worms per animal, while the hamster harbored 82.0±18.0 per animal. Interestingly, the number of male worms was less than thenumber of female ones on both of infected hamsters and infected hedgehog (Table 2).
No significant difference was observed in the distribution of worms among the mesenteric, portal and intrahepatic veins of the hedgehog (H = 5.7, p = 0.05) and the hamster (H = 3.5, p = 0.1) (data not shown). In both species, the mesenteric veins contained the greatest average number of schistosomes, followed by the portal and intrahepatic veins. However, no significant difference was recorded between the mean number of male and female worms in the three regions.
The distribution of schistosome eggs in animal tissues: Tissue digestion showed that the greater number of eggs was contained in the intestine, followed by the liver. These results were common to the hedgehog and the hamster. The difference observed was not significant (t = -1.2, p = 0.3) in hedgehog but significant (t = -7.9, p = 0.0) in the hamster. In the hedgehog, few eggs were found in the lung and no eggs were recorded in the spleen, while in the hamster few eggs were found in both organs. With respect to the total tissue egg count, a greater number of eggs were observed for the hamster than for the hedgehog (t = -7.9, p = 0.0) (Table 3).
|Table 2:||No. of worms recovered from the hedgehog and the hamster eight weeks post exposure to S. mansoni cercariae|
|*SmM: S. mansoni male, SmF: S. mansoni female. (μm3) *Significant at p<0.05|
|Table 3:||Distribution of S. mansoni eggs in the tissues of the hamsters and the hedgehogs|
|*Significant at p<0.05|
|Fig. 2(a-b):||Typical granulomatous reaction of the infected (a) Hamster and (b) Hedgehogs|
Granuloma volume measurements: To measure the granuloma volume of the 8 week-infected hedgehogs and hamsters, the mean diameter of at least 70 lesions from each species were determined and then the volume of each lesion was calculated to determine the granuloma volume. Figure 2 (a and b), the histo-pathological examination of the liver sections of the infected hamsters and the infected hedgehogs showed typical granulomatous reactions formed mainly of histocytes lymphocytes and eosinophils. Microscopic examination showed that there are no any calcified dead eggs in the liver tissues of the infected hedgehogs and most of eggs contain live meracidia and this observation could explain that these animals were recently infected with S. mansoni and they did not naturally infected. The results showed also that the granulomas volume of the infected hedgehogs measured 74.0±56.0 μm3 which was significantly smaller than those of the infected hamsters; 119.0±75.0 μm3.
Effect of S. mansoni infection on the biochemical parameters in liver tissues: The current study showed also that S. mansoni infection caused marked hepatic dysfunction in the hedgehogs as well as in the hamsters.
The liver enzymatic activities of cholinesterase, arginase, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were markedly reduced after 8 weeks post S. mansoni infection as compared to their counterparts. Furthermore, infection of hedgehogs and hamsters with S. mansoni led to decrease in the liver total protein contents and serum albumin, with an increase in the total lipids when compared with their normal counterparts (Table 4).
Although, Schistosoma mansoni shows a low specificity in relation to its vertebrate host choice, only few species are capable of developing the infection and allowing the parasite to complete its biological cycle in natural environment (Rey, 1993). The high susceptibility of different mammalian species to S. mansoni infection was reported (Barbosa, 1972; Costa-Silva et al., 2002; El-Naggar et al., 2011). So far, there are few data concerning about the potential role of the hedgehog, Hemiechinus auritus to act as a reservoir host for S. mansoni infection.
|Table 4:||Biochemical disorders in the liver of animals infected with S. mansoni infection|
|*Significant at p<0.05, activities and contents were expressed in: (1) μmol urea min-1 wet liver tissue (2) μmol SH min-1 wet liver tissue (3 and 4) μmol pyruvate min-1 wet liver tissue (5 and 6) mg g-1 wet liver tissue (7) mg (%)|
In this study, some aspects of S. mansoni biology in the hedgehogs were studied. Susceptible hosts to S. mansoni have been defined as those in which development of worms is complete, excretion of viable eggs in feces is not interrupted and parasite life cycle is maintained (Cioli et al., 1977).
The present study showed that no eggs were passed in the feces of the hedgehog, therefore, the hedgehog considers as a dead-end host for schistosomiasis. This indicates that some factors may influence the excretion of eggs with feces. These may include intrinsic or extrinsic factors to the parasites. Furthermore, the physiological state of the intestinal mucus can also cause the differences in egg excretion. Eggs deposited in mesenteric veins must migrate through the intestinal wall before being excreted with feces (Sene et al., 1996).
In this study, it has found that the enumeration of worm burden of the hedgehogs have low susceptibility to S. mansoni compared to the hamster. The worms were detected in the liver as well as in the mesenteric veins. These results were in agreement with Dias et al. (1978).
They reported that adult S. mansoni worms were found to be concentrated in portal and intra-hepatic veins in naturally infected Cavia aprea and Holochilus leucogaster. Similar findings were obtained from Nectomys squamipes naturally infected with S. mansoni (Silva et al., 1992).
In the hedge hog and the hamster, male and female worms were found to be evenly distributed in the mesenteric, portal and intrahepatic veins. These data disagreed with those found in N. squamipes, where a greater proportion of male worms were observed in the liver as well as the mesentery (Silva et al., 1992). Tissue digestion showed that in the hedgehogs and the hamsters, the largest number of eggs was contained in the intestine, followed by the liver. The total tissue eggs count was comparable in both animals. Quantitative oogram showed that a greater number of eggs were contained in the proximal portion of the intestine and that was in accordance with the results previously reported for N. rattus and N. squamipes (Silva et al., 1992).
Infection of the hedgehogs with S. mansoni caused marked pathological changes at histo-pathological and biochemical levels. The infection with S. mansoni caused the development of the characteristic granulomatous inflammatory reaction reported in permissive hosts; hamsters as well as the hedgehogs. Although of smaller volume of the granulomas were found in the liver hedgehogs, the cellular characteristic features of the inflammations are the same as those developed in the permissive host (Fig. 2). The reduction of granuloma diameter could be due to the reduction of type III procollagen which is responsible for the granulomas formation (Badawy et al., 1991).
As a result of S. mansoni infection, the liver is manifested by dramatic changes of the enzymatic activities. The current results showed that the values of the liver enzymatic activities of cholinesterase, arginase, ALT and AST, were markedly inhibited as a result of S. mansoni infection. These results were in agreement with the previous studies which showed that cholinesterase and arginase activities of the liver tissue homogenate showed a significant reduction in murine S. mansoni (Al-Sharkawi, 1996). With regard to transaminases, ALT is more specific for liver damage than AST (Wilkinson, 1976). The results showed also that the activities of ALT and AST in the liver tissue homogenate were reduced and this finding was in agreement with those found by Al-Sharkawi et al. (1989).
S. mansoni infection caused marked increase of the hepatic content of total lipids, which reflects the liver incapability for their mobilization. The liver contents of protein, serum albumin were also decreased while; the liver content of total lipids was elevated after the S. mansoni infection of hamsters and hedgehogs. Such increased water intake may explain the decrease in the total content of the liver protein reported in this study. However, the reduction of serum albumin is consistent and extends previous observations in experimentally infected monkeys (Bruce et al., 1963) and mice (Goodgame et al., 1978; Zakaria et al., 1981). In conclusion, the hedgehog showed low susceptibility to S. mansoni infection as compared to the hamster and such infection results in marked severe hepatic dysfunction. Furthermore, this host is not appropriate for schistosomiasis transmission in the field.
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