|Year : 2014 | Volume
| Issue : 2 | Page : 110-115
Heterophyid metacercariae in free living and farmed fish of El-Max Bay, West of Alexandria, Egypt
Mona H El Sayad1, Sahar A Abou Holw1, Omaima G Yassine2, Hend A El-Taweel PhD 1
1 Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt
2 Department of Biomedical Informatics and Medical Statistics, Medical Research Institute, Alexandria University, Alexandria, Egypt
|Date of Submission||24-May-2014|
|Date of Acceptance||02-Nov-2014|
|Date of Web Publication||19-Jan-2015|
Hend A El-Taweel
91 Ahmed Shawky Street, (13/7), Moustafa Kamel, 21523, Alexandria
Source of Support: None, Conflict of Interest: None
The role of fish living freely in their natural habitats in the transmission of fish-borne trematodes is well recognized. Moreover, the role played by aquaculture fish has also gained great attention in the last few years.
To investigate the rate, density, distribution of infection, and infectivity of heterophyid metacercariae in free living and farmed fish collected from El-Max Bay, a Mediterranean coastal bay in Egypt. The influence of freezing duration on the infectivity of the detected metacercariae was also evaluated.
Materials and methods
Tilapia nilotica and Mugil cephalus from both habitats were examined for encysted heterophyid metacercariae using a compression method. The density of infection was estimated by the number of metacercariae per gram of trunk tissue following artificial digestion. The distribution of infection was studied in snips taken from the head, gills, trunk, viscera, and tail. Infectivity of the collected metacercariae was tested in rats. The susceptibility of metacercariae to freezing was evaluated by assessment of their infectivity to rats after they were kept frozen at −15°C for 4, 7, and 14 days.
Rates of infection with heterophyid metacercariae ranged from 11 to 23% in the different groups of fish. Free living fish had a significantly higher rate of infection and/or density as well as higher infectivity of metacercariae than farmed fish. Higher metacercarial density was observed in the trunk and viscera of the studied fish compared with the head, tail, and gills. Infectivity of the detected metacercariae decreased gradually with increasing duration of freezing.
Both free living and farmed fish can transmit Heterophyes parasites, the former being somewhat more important. The potential risk of human infection is considered to be high. Freezing for 2 weeks is an effective means of inactivating the parasite. Our results underscore the need to raise awareness among public health agencies, consumers, and aquaculture managers of the measures needed to reduce transmission of this intestinal fluke.
Keywords: Egypt, farmed fish, Heterophyes spp., metacercariae, Mugil cephalus, Tilapia nilotica
|How to cite this article:|
El Sayad MH, Abou Holw SA, Yassine OG, El-Taweel HA. Heterophyid metacercariae in free living and farmed fish of El-Max Bay, West of Alexandria, Egypt. Parasitol United J 2014;7:110-5
|How to cite this URL:|
El Sayad MH, Abou Holw SA, Yassine OG, El-Taweel HA. Heterophyid metacercariae in free living and farmed fish of El-Max Bay, West of Alexandria, Egypt. Parasitol United J [serial online] 2014 [cited 2017 Dec 17];7:110-5. Available from: http://www.new.puj.eg.net/text.asp?2014/7/2/110/149560
| Introduction|| |
Egypt has large areas of fresh, brackish, and marine water bodies suitable for fishing. Moreover, the climatic conditions are optimum for fish farming, with high growth rates for the fish that are raised, which constitute a cheap source of animal protein for Egyptians , . Fish are a potential source of several pathogens to humans  . Although studies have long documented the importance of wild-caught fish in the transmission of fish-borne pathogens, the role played by aquaculture fish has gained considerable attention only in the last few years , .
Heterophyiasis, caused by minute intestinal digenetic flukes, is one of the fish-transmitted zoonotic diseases. Although most human infections are asymptomatic or pass unrecognized, heavy infection may cause mucosal damage and some gastrointestinal symptoms. Eggs rarely reach ectopic locations, causing serious damage , . Transmission of heterophyiasis occurs in water bodies where the mollusc and fish intermediate hosts coexist. Metacercariae present in fish are the infective stage to humans, dogs, and cats, the definitive hosts , . Heterophyiasis is endemic in some areas of the Nile Delta, where the habit of consuming raw or inadequately cooked fish favors its transmission ,, .
Mullets and Tilapia fish are implicated in the transmission of heterophyiasis in Egypt  . Mullets are found in fresh, brackish, and marine water bodies of tropical and subtropical areas. The flat head gray mullet, Mugil cephalus, can be polycultured successfully with many other fish including Tilapia  . In Egypt, it is dried and salted in a special way to make a dish known as Feseekh. The freshwater fish Tilapia nilotica is of high economic value because of its rapid growth rate. It tolerates a wide range of environmental conditions and can be farmed easily  .
Control of fish-transmitted zoonoses depends mostly on adopting methods that render fish metacercariae noninfectious, such as elimination of the snail host and avoiding contamination of water bodies by egg-containing excreta  . The latter approach is often hampered by water runoff from the surrounding environment  .
Assessment of the prevalence, density, and infectivity of encysted heterophyid metacercariae in fish in a certain area provides information on the risk of heterophyiasis to humans and can guide measures for its prevention. The present work investigated the detection rate of encysted heterophyid metacercariae in T. nilotica and M. cephalus collected from natural brackish water and aquaculture in a coastal area west of Alexandria. The detected metacercariae were tested for their density, distribution of infection, infectivity, and susceptibility to freezing.
| Materials and methods|| |
Type of the study
Descriptive analytical study.
Fish were collected from El-Max Bay in the west of Alexandria City. This Mediterranean coastal bay extends between El-Agamy headland to the west and the Western Harbor to the east, with a surface area of about 19.4 km 2  . El-Max Bay water is brackish as its salinity is influenced by the rate of water exchange with the adjoining sea and the amount of water received from Lake Mariout through El-Umum Drain  . Aquaculture is practiced in El-Max fish farm, which is one of the most important fish farms in Alexandria. This farm receives water from the El-Umum drain through a canal called El-Moghazy and from three wells drilled inside the farm. The conditions of the farm are suitable for aquaculture of T. nilotica and M. cephalus fish  .
Fish collection and detection of heterophyid metacercariae
Fish were collected from El-Max Bay during the years 2011-2012. A total of 463 fish comprised the study sample. There were 180 T. nilotica and 120 M. cephalus living freely in natural brackish water (wild-caught fish) and 72 T. nilotica and 91 M. cephalus from man-made reservoirs (aquaculture or farmed fish). Fish were transported alive in plastic bags to the laboratory, where the fish body was divided into head, gills, trunk, viscera, and tail. All body parts were compressed between two microscopic glass slides and screened for the presence of heterophyid metacercariae  .
Density and distribution of metacercariae
The density of metacercariae in infected fish was determined using a digestion method: 10 g of the trunk tissue was digested for 1 h at 37°C in an acidified pepsin solution and then filtered through a mesh to remove large debris. The filtrate was then left to sediment and the precipitate was examined microscopically for metacercariae  . The density was estimated by the number of metacercariae/g of trunk tissue. The distribution of metacercariae in the different fish body parts (head, gills, trunk, viscera, and tail) was studied in 10 fish of each group. Three snips (~1 g each) were taken from each part and examined using the compression method described above. The metacercariae detected were counted and the average number/g was recorded for each body part  .
Infectivity of metacercariae
Metacercariae collected from fish were used to infect four groups of rats: metacercariae from T. nilotica of natural water (group I) and aquaculture (group II), and metacercariae from M. cephalus of natural water (group III) and aquaculture (group IV). Each group included 10 rats. Each rat was orally inoculated with 100 fresh metacercariae using an esophageal syringe  . Rats were killed 21 days postinfection (PI). The small intestines were excised, opened longitudinally, and washed with warm saline in petri dishes. The flukes were collected and counted. Some flukes were washed several times with warm saline, fixed in 10% neutral-buffered formalin, and stained with acetocarmine to study their morphological characteristics , .
Effect of freezing duration on metacercarial infectivity
Some of the metacercariae collected from different sources were kept frozen at −15°C for 4, 7, or 14 days. Three groups, each of 10 rats, were infected orally with the frozen metacercariae; each group corresponded to one duration period of freezing. The fourth control group was orally inoculated with unfrozen metacercariae. Rats were killed 21 days PI. The effect of different freezing durations on metacercarial infectivity was compared by the percentages of metacercariae that had developed to adult worms 3 weeks PI with 100 metacercariae.
The study was approved by the Ethical Committee, Medical Research Institute, Alexandria University, Egypt.
Data analysis was carried out using the statistical package for social sciences (SPSS version 17.0, 2006; SPSS Inc., Chicago, Illinois, USA). Normality of quantitative variables was tested using the Kolmogrov-Smirnov test. Normally distributed quantitative variables were described as mean and SD, whereas non-normally distributed variables were described as median and range. Comparison between the means of two different groups was carried out using the independent t-test, whereas comparisons between the means in the different fish species were carried out using the independent analysis of variance test, followed by a pairwise comparison using Tukey's HSD (Honestly Significant Difference) test. Comparison between the medians in the same species of fish was performed using Friedman's test, followed by the Wilcoxon signed rank test for pairwise comparisons. Comparisons between different percentages were performed using the χ2 -test. The χ2 for trend was used to test for a linear trend in percentages. All tests were two sided and the significance was set at 0.05, except for pairwise comparisons, where the corrected significance level was computed according to the number of comparisons made (Bonferroni's correction).
| Results|| |
A higher rate of metacercarial infection was recorded among free living fish (23.3% for T. nilotica and 19.2% for M. cephalus) compared with aquaculture fish (11.2 and 13.2%, respectively). This difference was statistically significant only for T. nilotica (χ2 = 4.09, P < 0.05) ([Table 1]).The metacercarial density in trunk tissue was also significantly higher among free living compared with aquaculture fish, and this applied to both T. nilotica (132 ± 18.2 vs. 14 ± 3.3/g) and M. cephalus (93 ± 14.9 vs. 9 ± 2.9/g) ([Table 2]).
|Table 1 Prevalence of metacercarial infection in Tilapia nilotica and Mugil cephalus collected from the El-Mex Bay by the source of collection during the period 2011– 2012|
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|Table 2 Metacercarial density in fish examined from the El-Max Bay by the source of collection during the period 2011– 2012|
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For all the groups studied, the distribution of metacercariae/g tissue was significantly different between the fish body parts. The highest numbers were in the trunk, followed by the viscera, then the head and tail, and finally the gills ([Figure 1]).
|Figure 1 Number of heterophyid metacercariae/g tissue of different body parts of Tilapia nilotica from a natural habitat (a ) and a man-made reservoir (b) and Mugil cephalus from a natural habitat (c) and a manmade reservoir (d). *Significant difference compared with the head, °significant difference compared with the gill, •significant difference compared with the trunk, ◊significant difference compared with the viscera, ¶significant difference compared with the tail.|
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The infectivity of metacercariae obtained from T. nilotica and M. cephalus was confirmed by positive infection of all studied rats. The collected flukes appeared as pear-shaped bodies measuring 1-2.5 mm in length. They had a small oral sucker, a large median ventral sucker, and a submedian genital sucker armed with many rodlets. Two side-by-side testes were observed in the broad rounded posterior end. The flukes were identified as Heterophyes heterophyes  .
A statistical comparison of the mean number of the recovered worms showed that metacercarial infectivity was generally higher among Tilapia spp. than Mugil spp. For each species, infectivity was significantly higher for metcercariae of free living than farmed fish ([Table 3]).
|Table 3 Infectivity of metacercariae collected from Tilapia nilotica and Mugil cephalus fish of the El-Max Bay natural source and aquaculture|
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[Figure 2] shows the effect of different durations of freezing on metacercarial infectivity as assessed by the percentage of metacercariae that had developed to adult flukes in the intestine of rats inoculated with 100 metacercariaea compared with controls. The metacercarial infectivity rate showed a statistically significant decrease with an increase in the duration of freezing at −15°C. The percentages of worms recovered decreased from 36% in the controls to 20, 5, and 0% after freezing for 4, 7, and 14 days, respectively (χ2 trend = 58.53, P < 0.001).
|Figure 2 Effect of three different freezing duration periods on metacercarial infectivity evaluated by percent worm recovery in three rat groups (10 each) 3 weeks after inoculation of each rat with 100 metacercariae kept frozen at −15°C compared with a control group inoculated with unfrozen metace rcariae.|
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| Discussion|| |
Heterophyiasis is prevalent in humans in many countries in the Middle East and Far East. Fish act as the first intermediate host and the habit of consuming raw or inadequately cooked or salted fish is an important determinant of human infection , . In the present study, heterophyid metacercariae were detected in about one-fifth of T. nilotica and M. cephalus fish living freely in El-Max Bay west of Alexandria. Infection rates very similar to those observed in the present work have been reported in different fish species collected from other coastal areas in Egypt , . However, a markedly high rate of infection (95%) was reported in Tilapia fish collected from the Ismailia freshwater canal to the east of the Nile Delta  . Conditions favorable for the transmission of fish-borne trematodes include lack of awareness or ignorance of infection by public health authorities, poor sanitary conditions, and waste disposal in water bodies , . Such situations prevail in some localities in Egypt.
Variation in fish infection rates from one study to another is related to local conditions that sustain the parasite life cycle. In the area chosen for the present study, heterophyid eggs are probably carried in agricultural drainage water discharged into the bay through El-Umum drain  . In addition, domestic animals and fish-eating birds, which act as definitive hosts, may play a role in maintaining the life cycle of the fluke in this area  . However, El-Max Bay is located in an industrial area and receives a heavy load of industrial wastes and pollutants  . This may reduce the swimming rate and the longevity of cercariae, resulting in failure of transmission  . The type of water, whether fresh or brackish, also appears to play an important role; a higher rate of infection with heterophyid parasites has been reported previously in fresh compared with brackish water fish  .
The expansion of modern aquaculture began in Egypt two decades ago and particularly over the last few years. Now, fish farming represents an important affordable source of fish for the majority of Egyptians  . In fish farms, the passage of infected snails and/or cercariae with the feeding water, the use of human and domestic animal excreta as fertilizer, and sewage runoff allow for completion of the life cycle of heterophyid parasites , . Our results showed the presence of metacercariae in farmed fish, but with a significantly lower metacercarial density and infectivity in both T. nilotica and M. cephalus, and a lower infection rate in T. nilotica compared with the corresponding free living species. A higher risk of infection with trematode parasites in wild-caught compared with farmed fish has been reported previously  . This may be attributed to the greater abundance of the snail intermediate host in the natural habitat compared with man-made reservoirs, where the use of nets, cages, or other barriers keep snails away; thus, the proportion of cercariae reaching fish may be lower. The probability of infection of farmed fish may also be low because of regular repeated harvesting, which reduces the duration of fish exposure to cercariae. In addition, the wide use of antimicrobials and other chemotherapeutants in aquacultures to enhance fish production might influence the development and infectivity of the different life cycle stages of the parasite  .
In the present study, significant differences in the distribution of metacercariae among different fish body parts were found, with higher values in the trunk and viscera than in the head, tail, and gills. A previous study reported that the highest distribution of heterophyid metacercariae in Tilapia fish was in the head region  . Other studies found that the posterior third are highly affected with metacercariae compared with the head region , . Moreover, recently, it was recorded that the distribution of metacercariae also varies among the visceral organs. Portes Santos et al.  reported a higher prevalence of metacercariae in the intestine and liver of Mugil liza compared with the stomach, whereas infection in the gall bladder and gonads was relatively low  .
A high level of metaceracrial infectivity was confirmed in the present study by the ability of the metacercariae collected from different fish groups to initiate infection in all studied rats. This indicates the risk of human infection posed by consumption of these species of fish. The higher infectivity of metacercariae collected from T. nilotica compared with M. cephalus suggests that the former is more supportive for parasite development; the factors beyond this observation deserve further elucidation.
In the present study, the percentage of H. heterophyes worms recovered from rats inoculated with 100 fresh unfrozen metacercariae from different sources was only 36%. Previous studies showed that, in experimental infection, the recovery rate of adult heterophyid flukes depends on the species of the experimental host as well as the species of the parasite. Li et al.  reported adult Metagonimus yokogawai recovery rates of 6% in gerbils, 23% in rats, and 75% in hamsters  . Elsheikha and Elshazly  reported that the fluke recovery rate in puppies was 19% for H. heterophyes and 9% for Stictodora tridactyla  .
Methods of fish processing before human consumption influence metacercarial viability and infectivity  . On evaluating susceptibility to freezing, the present study showed that the metacercarial infectivity rate decreased with increasing days of freezing at −15°C and that infectivity was totally lost after 2 weeks of freezing. The WHO has recommended freezing as an effective means to reduce the risk of fish-born trematodiasis  . Several studies on the effect of the freezing temperature and the time of exposure to metacercarial infectivity can be found in the literature ,, . It was suggested that the duration for which the frozen metacercariae survive depends on the size of metacercariae as well as the species of fish from which they were collected  .
| Conclusion|| |
The present study showed the occurrence of heterophyid metacercariae in two commonly consumed fish species collected from the El-Max area in northern Egypt. Both free living and cultured fish can transmit Heterophyes parasites, although the former is somewhat more important in view of the higher infection rate and/or density as well as infectivity of metacercariae. The potential risk of human infection is considered to be high. Raw fish freezing for 2 weeks is an effective means of eliminating this risk. Our results underscore the need to raise the awareness of public health agencies, consumers, and aquaculture operators and managers on the transmission of this intestinal fluke. Measures should be implemented to keep natural and man-made fish habitats free from raw sewage.
| Acknowledgements|| |
The authors are grateful for the help and support provided by Professor Awatef Mohamed Ali, Zoology Department, Faculty of Science, Alexandria University, Egypt, in fish collection.
| Author's contribution|| |
M.H. El-Sayad proposed the study topic, participated in the practical work, and reviewed the literature. S.A. Abou Holw and H.A. El-Taweel participated in the design, conception, and implementation of work, interpretation of the results, and writing the manuscript. O.G. Yassine carried out the statistical analyses and data presentation.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]