Abstract: Background and Objective: Fish serve as appropriate hosts for several parasitic helminths, particularly digeneans. In this study, presence of encysted metacercariae (EMC) of such helminths and their effects on the quality of freshwater fish was examined. Trematodes recovered from puppies experimentally infected with EMC (in Oreochromis niloticus (O. niloticus) and Clarias gariepinus (C. gariepinus) fish) were identified and chemical alterations in fish were evaluated. Materials and Methods: A total of 200 fish (100 O. niloticus and 100 C. gariepinus) were collected from markets in Kafrelsheikh, Egypt. Collected fresh specimens underwent parasitological examination searching for metacercariae as well as biochemical tests were done. Experimental infection, using the recovered metacercariae, for puppies was applied to identify adult flukes. Chemical parameters reflecting fish freshness were used. Among those, moisture, protein contents, oil contents, ash, pH, total volatile nitrogen, thiobarbituric acid, peroxide value and free fatty acids were evaluated. Results: The prevalence of digenean EMC infection in both O. niloticus and C. gariepinus was 24.0 and 30.0%, respectively, predominantly in dorsal regions. Head, tail and anal regions had variable degrees of infection. Heterophyids [Heterophyes heterophyes (H. heterophyes) and Heterophyes aequalis (H. aequalis)] and haplorchids [Haplorchis pumilio (H. pumilio)] were recovered from O. niloticus, while cyathocotylids [Prohemistomum vivax (P. vivax) and Mesostephanus spp.] were found in both species. The chemical composition of fish was significantly (p<0.01) affected. Heavy infection induced a significant decrease in protein, oil and ash contents with a significant (p<0.01) increase in the total volatile nitrogen, thiobarbituric acid number, peroxide value and free fatty acids. Moreover, a non-significant difference in moisture contents and the pH degree of muscles was found. Conclusion: The recovery of adult digeneans from experimentally infected puppies reflected that eaten fish harboured encysted metacercariae. Chemical composition of infected fish had some alterations. Accordingly, parasitologists, ichthyologists, veterinarians and authorities must greatly consider the digeneans-fish relationship via the proper control of eating raw/inadequately cooked fish which assume reduced nutritional values.
INTRODUCTION
Fish production is one of the most important food industries due to the rapid growth cycle and the low price1. As an excellent source of quality protein and low saturated fats, fish possess great nutritive values and so act as important healthy human diet. Various diseases, including parasitic infections, pose a threat to fish culture resulting in significant losses in the fish industry2. The harmful effects of parasites greatly vary, depending on the parasite proliferation, the degree of adaptation developed within the host, the size of the host and its health status3.
The presence of metacercariae results in a low weight gain, high mortality, in market ability of the infected fish4. In addition to economic losses, several parasites are of zoonotic importance. Eating raw or improperly cooked fish is the main source of infection in humans, especially for people who live near the water sources5,6.
It is known that about 18 million people around the world became infected via the ingestion of infective metacercariae in raw fish, with more are at risk. Many species of fish act as the second intermediate host for parasitic digenean EMC causing infection to humans and to other fish eaters1,7. EMC cause superficial necrosis in the intestinal mucosa of humans and the ingestion of inadequately cooked fish can lead to severe visceral pain, abdominal discomfort, intermittent bloody diarrhea and colic, especially tilapia species8. Parasitic infection in fish is so detrimental to the fish industry, lowering the quality and quantity of production and hence the economic value of fish9.
Accordingly, the present study aimed to study the prevalence of EMC in some fresh water fish species, to identify the parasites by the experimental infection of puppies and to assess fish quality by analyzing fish muscle for chemical and spoilage indicator parameters according to the degree of parasitic infection.
MATERIALS AND METHODS
Samples collection: A total of 200 fish, O. niloticus and C. gariepinus (100 fish each), were collected at weekly intervals from different fish markets in Kafrelsheikh province (SediGhazy, Billa and Kafrelsheikh) (coordinates: 31.3°N 30.93°E), Egypt during spring, 2015. The fish were collected, by fishermen from wild canals and examined freshly as soon as they collected. Fish were packed in polyethylene bags and stored with ice bags for 1 h till transported to the laboratory. Collected samples of O. niloticus were brought to the laboratory in a fresh killed state. Then, C. gariepinus specimens were brought alive. Each species arrived separately at the laboratory. The fish were dissected separately exposing their muscles.
Parasitological examination Detection of metacercariae: Collected fish specimens were macroscopically examined by the naked eye or by hand-held magnifying lens. A small specimen of each fish muscle obtained from several body regions was compressed between 2 glass slides examined for the presence of encysted metacercariae10. The examined sites were the head region, trunk and the base of all fins (dorsal, tail and anal). The intensity of infection per gram was divided into 3 categories: Low infection (<3 EMC), moderate infection (3-7 EMC) and high infection (>7 EMC).
Experimental infection: Forty five puppies aged 2 weeks were taken from Laboratory Animal and Pets House conducted in Faculty of Veterinary Medicine, Kafrelsheikh University and divided into 3 equal groups. Periodic examination of fecal samples showed that dogs were parasitic free. The first group was infected with approximately 1000 EMC obtained from infected muscles of the O. niloticus. The second group was infected with the same dose in muscles of C. gariepinus. The third group was left as control non-infected. All puppies were reared on dry bread, some vegetables, some protein sources and clean water. All institutional, ethical and animal care guidelines were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
Recovery of adult flukes: Daily fecal examination was done for the detection of parasitic infection. Concomitantly, puppies were sacrificed 10, 20 and 30 days post infection (dpi) to obtain the adult flukes. The intact worms were collected and identified according to Yamaguti11.
Chemical examination of fish samples Determination of moisture percentage: According to the method described by AOAC12, the moisture content was calculated through the following equation:
Determination of protein percentage: In Kjeldahl flask, the correlation between crude proteins and nitrogen was found as follows12:
Crude protein = Nitrogen×6.25
Determination of oil percentage: In Soxhlet apparatus, fat contents were extracted according the method described by AOAC12.
Determination of ash percentage: In a porcelain crucible of known weight, the ash percentage was calculated according the following formula12:
Percentage of Ash = Crucible with ash-empty crucible
Determination of pH: The pH value was determined by using an electrical pH meter (Bye model 6020, USA)13.
Determination of total volatile nitrogen (TVN) (mg%): The technique applied for determination of total volatile nitrogen (TVN) was recommended by FAO14 as follows:
TVN/l00 g = 26.88×(2-T2)
Where:| T2 | = | Volume of NaOH consumed in the titration |
Determination of thiobarbituric acid number (TBA) (mg kg–1): Following the method adopted for estimation of TBA was calculated according to Vyncke15:
Concentration of malondialdehyde (mg kg–1) = 0.016+2.872X
Where:| X | = | Absorbance |
Determination of peroxide value: In Erlenmeyer flask, after titration with sodium thiosulfate solution, the peroxide value was calculated according to the method described by Asakawa and Matsushita16.
The peroxide value (PV) for all samples was calculated according to the following formula:
| V1 | = | Volume of thiosulfate solution required to titrate the sample (mL) |
| V0 | = | Volume of thiosulfate solution required to titrate the blank |
| T | = | Titre of the sodium thiosulfate solution (normality) |
| m | = | Weight of sample (g) |
Determination of free fatty acids (FFA): Lipid was extracted by the use of the method assembled by Folch et al.17. The lipid extract of the sample was titrated with sodium hydroxide to a violet end point18. Cresol purple has been proposed to increase the sensitivity of the method and has been used for the titration of lipid fish extracts19. Accordingly, FFA values were recorded as the percentage of oleic acid as follows:
Statistical analysis: The experimental design was randomly evaluated with five replicates for each species. The obtained data were subjected to the analysis of variance (one way ANOVA)20. Then, the treatment means were compared by Duncan’s multiple range test21 among the tested groups. All statistical analyses were performed using the analysis of variance technique ("MSTATC" computer software package). Values were considered significant at p<0.01.
RESULTS
In the present investigation, it has been found that 24 (24.0%) out of 100 O. niloticus and 30 (30.0%) out of 100 C. gariepinus harboured digenean encysted metacercariae (Table 1 and Fig. 1). Among infected fish, it was shown that the dorsal regions were the highest infected (in all infected fish), trunks were moderately infected (66.7% in O. niloticus and 100% in C. gariepinus) and the lowest infection rates were found in head, the anal (66.7% in both) and the tail (66.7% in O. niloticus and 83.3% in C. gariepinus) regions (Table 2). As the percentage reflected the number of infected fish, in Table 2, number of fish infected was similar in trunk, tail and the anal regions [each 16 (66.7%)] regardless the intensity of infection.
The current study revealed that adult flukes recovered from puppies experimentally infected with EMC in O. niloticus were H. heterophyes, H. aequalis and H. pumilio. Meanwhile, adult worms recovered from puppies experimentally infected with EMC in C. gariepinus were Mesostephanus spp. and Prohemistomum vivax (Table 3 and Fig. 2).
The pH degree of fish muscles was evaluated as reliable freshness indicator. The revealed low muscle pH in negatively infected fish reflected a good physical status, although there was a slight non-significant (p<0.01) elevation in pH in moderately and heavily infected fish. The condition was affected by storage time or processing. Moreover, moisture content had a non-significant (p<0.01) difference in both fish species.
| Table 1: | Mean intensity of infection with encysted metacercariae (EMC) in Oreochromis niloticus and Clarias gariepinus |
| Table 2: | Prevalence and intensity of infection with EMC in Oreochromis niloticus and Clarias gariepinus |
Int: Intensity of infection, Low: Infection with lower than 3 EMC, Moderate: Infection with 3-7 EMC and High: Infection with more than 7 EMC |
|
| Table 3: | Experimental infection of puppies with encysted metacercariae (1000 EMC/puppy) |
dpi: Days post infection |
|
| Fig. 1(a-d): | Encysted metacercariae found in muscles of infected Oreochromis niloticus and Clarias gariepinus, (a) Heterophyids. (b) Haplorchis pumilio, (c) Prohemistomum vivax and (d) Mesostephanus spp. |
Scale bar = 100 μm |
|
| Fig. 2(a-e): | Adult trematodes recovered from infected puppies, (a) Heterophyes heterophyes, (b) Heterophyes aequalis, (c) Haplorchis pumilio, (d) Prohemistomum vivax and (e) Mesostephanus spp. |
Scale bar= 100 μm |
|
| Table 4: | Chemical alterations in flesh of Oreochromis niloticus as a result of encysted metacercariae |
Ns: Non-significant and **Significant p<0.01 |
|
| Table 5: | Chemical alterations in flesh of Clarias gariepinus as a result of encysted metacercariae |
Ns: Non-significant and **Significant p<0.01 |
|
Oppositely, there was a significant (p<0.01) decrease of protein content in heavily infected O. niloticus and C. gariepinus, respectively (19.9-18.2 and 18.6-17.1%) compared to the negatively infected fish. Significantly low percentages of oil and ash were recorded in moderate and heavily infected fish compared to uninfected fish (Table 4, 5). A significant (p<0.01) elevation in peroxide value (PV), thiobarbituric acid (TBA) and free fatty acids (FFA) was determined in fishes. Meanwhile, total volatile nitrogen (TVN) significantly (p<0.01) increased in moderate and heavily infected fishes.
DISCUSSION
The current investigation elucidated the prevalence of infection of both O. niloticus and C. gariepinus with digenean encysted metacercariae. In the first, the infection rate was 24.0%. Such prevalence was closely related to that obtained by Mahmoud 22, who reported 21% infection rate. Oppositely, it was lower than those given by Taher23 in Assuit (78.25%), Reda et al.24 in Sharkia (80.30%), Ibrahim and Soliman25 in Ismailia (95.3%), Lubna et al.26 in Northern Egypt (42%) and Hegazi et al.27 in Dakahlia (42.6%). In C. gariepinus, the prevalence was 30.0%. A similar finding was recorded by Shalaby28 in Cairo (39.3%) but it is significantly lower than that recorded by Eldaly et al.29, who reported prevalence of 87.5% in Damietta. The distribution of snails facilitates the completion of life cycle of such flukes. The discrepancies in prevalence might be due to the different topographic areas investigated.
Regarding tissues affected, it has been found that the dorsal regions were the most infected. Such finding was similar to that given by El-Seify et al.1. On the other hand, Saba30 found that the highest infected regions were the tail and caudal parts. The variation in the preferred infected sites might be due to species of fish and the genetic variation of the EMC31.
The present study revealed that adult heterophils were recovered from puppies infected with EMC obtained from O. niloticus. Elsheikha and Elshazly32 recovered heterophyids in puppies fed on EMC from infected fish muscles. El-Seify et al.1 and Elday et al.29 reported the same flukes in rats and mice. Such finding agreed with that given by El-Seify and Nabih33 and Mousa et al.34, who collected similar worms from stray dogs in Giza and Beni-Suef, respectively. Moreover, Mesostephanus spp. and Prohemistomum vivax were revealed from puppies eaten infected C. gariepinus. In Saudi Arabia, Khalil et al.35 recovered adult Heterophyes heterophyes and Haplorchis pumilio in kittens fed on EMC in muscles of fish.
Since fish act as carriers for many parasitic zoonosis, parasitologists have a long-standing interest in the understanding and characterization of the specific sites that parasites occupy within individual hosts36. Unfortunately, scarce studies are available on the effects of EMC on fish quality and consequently, on the effect of the parasites on the nutritional value of fish for humans. The O. niloticus and C. gariepinus act as a second intermediate host for some parasites, H. heterophyes, H. aequalis, H. pumilio, P. vivax and Mesostephanus spp. Those parasites play an important role in determining the health status of fish and their existence may lead to both economic losses and public health problems.
Previous literature revealed that there was a remarkable correlation between pH and fish freshness, suggesting that it could be a good and effective tool for the analysis and evaluation of fish freshness. It may be superior to the sensory evaluation with many inherent uncertainties37. Due to complexity of lipid oxidation, multiple spoilage parameters were used. Accordingly, elevation in peroxide value, thiobarbituric acid and free fatty acids was determined in fish as a result of infection indicating a marked decrease in fish quality, which in turn, reflect on the human health. Products of lipid oxidation are associated with aging, membrane damage, heart disease and cancer38. Almost all amino acids react with the products of oxidized lipids, decreasing the digestive metabolizing of protein, amino acids and fats and thus, may affect the weight gain39. Meanwhile, increased total volatile nitrogen in moderate and heavily infected fish indicating the release of trimethylamine and ammonia due to bacterial contamination and the decomposition of amino acids, rendering fish proteins spoiled and unfit for human consumption40. The oxidation of lipids not only produces rancid odors and flavors but also decreases the nutritional quality and safety of fish by the formation of secondary products 41. The rancidity of O. niloticus and C. gariepinus was determined by the PV test, indicating an early stage of oil oxidation, while the TBA and FFA assessed the extent of oxidative deterioration in fish. Increased values gave rise to rancid odor and flavor limiting the storage life of such species. The current study revealed that the level of EMC parasitic infection had a negative impact on all biochemical parameters except for the moisture content, in the two fish species. Furthermore, the increased spoilage indicator parameters pose to the potential negative impact on human health. In both marine and freshwater fishes, metacercariae of the family Heterophyidae are non-specific in their affinity to definitive hosts. They cause infections in domestic animals and in humans consuming raw or incompletely cooked fish42. Belizario et al.43 and Kim et al.44 recorded that H. heterophyes is endemic in the Nile Delta of Egypt. Heterophyid infections result in a reduction in fishery yields, with huge economic losses. Heterophyid metacercariae may be found in different parts of the fish body, gills, fins, scales, muscles and visceral organs45, causing severe gill damage and the decrease in respiratory tolerance, simultaneously, raises mortality in pond-based fish in the subtropics and tropics46. The main clinical symptoms of heterophyiasis in humans are diarrhea and colicky abdominal pain. However, migration of eggs to extra intestinal sites such as heart, liver and brain may occur resulting in potentially fatal consequences43. It is noteworthy that Prohemistomum vivax, which has rarely been recorded in humans, may cause death47. Strict hygienic precautions must be considered to assume fish more or less parasitic (particularly helminths) free. In such concern, eradication of snails is urgently demanded to close the life cycle of digenean trematodes. Further investigations on several species of fish, either fresh water or marine, using both traditional and molecular tools must be done to minimize hazard effects of consumed fish.
CONCLUSION
This study elucidated the existence of digenean encysted metacercariae in fish collected from different markets in Kafrelsheikh province, Egypt. Experimental infection of puppies with EMC-infected O. niloticus revealed H. heterophyes, H. aequalis and H. pumilio. Adult worms recovered from experimentally infected puppies infected with EMC in C. gariepinus were Mesostephanus spp. and P. vivax. Chemical indicators of affected fish revealed a significant reduction in protein contents as well as higher levels of peroxide value, thiobarbituric acid and free fatty acids. Further studies should be considered evaluating effects of other parasites including monogeneans, crustaceans and protozoa on the chemical composition of various species of fish.
SIGNIFICANCE STATEMENT
This study discovers helminths, particularly digenean trematodes, infecting O. niloticus and C. gariepinus fish in Kafrelsheikh, Egypt that can be beneficial for determining the hazard effects of consuming such fish through measuring some chemical parameters. This study will help the researchers to uncover the critical areas of fish-helminths relationship that many researchers were not able to explore. Thus, a new theory on achieving food (fish) safety may be arrived at.