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Effects of fermentation time and low temperature during the production process of Thai pickled fish (pla-som) on the viability and infectivity of Opisthorchis viverrini metacercariae
International Journal of Food Microbiology 218 (2016) 1–5
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Effects of fermentation time and low temperature during the production process of Thai pickled fish (pla-som) on the viability and infectivity of Opisthorchis viverrini metacercariae Sudarat Onsurathum a,b, Porntip Pinlaor b,c, Ornuma Haonon a,b, Apisit Chaidee a,b, Lakhanawan Charoensuk a,b, Kitti Intuyod b,d, Thidarut Boonmars a,b, Porntip Laummaunwai a, Somchai Pinlaor a,b,⁎ a
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, Thailand c Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand d Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e
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Article history: Received 27 April 2015 Received in revised form 27 October 2015 Accepted 8 November 2015 Available online 10 November 2015 Keywords: Opisthorchis viverrini Metacercariae Freshwater cyprinid fish Pla-som fermentation Food safety
a b s t r a c t Contamination of a popular fermented fish dish, pla-som, by Opisthorchis viverrini metacercariae (OVMC) is a possible cause of carcinogenic liver fluke infection in Thailand. Affected individuals are at risk of bile duct cancer, which is a major health problem for people in the Greater Mekong Subregion. In order to investigate concerns about food safety, we studied the effects of fermentation time and low temperature on the viability and infectivity of OVMC during the pla-som production process. Pla-som was prepared at room temperature for up to 1 week in duplicate experiments using cyprinid freshwater fish obtained from an O. viverrini-endemic area. OVMC were then isolated and identified under a stereomicroscope. Complete and viable OVMC were found on days 1–4 of fermentation, while their morphology was degenerated thereafter. After OVMC were fed to hamsters, the percentage of the worm recovery after 1 to 2 months of infection was 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively. In order to measure the effect of low temperature on OVMC, fish were kept in a refrigerator (4 °C) for up to five days and then subsequently fermented for three days. In fish stored in a refrigerator for 1 and 2 days, viable OVMC were clearly observed and were able to infect hamsters, a worm-recovery percentage of 3.3% and 12.7%, respectively. By contrast, in pla-som prepared from fish stored for 3 to 5 days, OVMC were degenerated and could not infect the host. In conclusion, pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing can prevent O. viverrini infection. This study may increase awareness of fermented-fish dish preparation to prevent liver fluke infection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Eating of semi-cooked or fermented fish dishes is associated with fish-borne trematode (FBT) infection because these foods usually contain metacercariae, an infective stage of trematode parasites, including minute intestinal and liver flukes (Chai et al., 2005; Keiser and Utzinger, 2009; Ziegler et al., 2013). Human small liver flukes consist of Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis. O. felineus is found in some parts of the European Union and the former Soviet Union, C. sinensis is found in eastern Asia, while O. viverrini is found in the Greater Mekong Subregion: Vietnam, Laos, Myanmar, Cambodia and Thailand (IARC, 2012). In Thailand, at least 6 million people are currently infected with the human liver fluke O. viverrini (Jongsuksuntigul and Imsomboon, 2003; Sithithaworn et al., 2012). ⁎ Corresponding author at: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (S. Pinlaor).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.11.001 0168-1605/© 2015 Elsevier B.V. All rights reserved.
Infection with O. viverrini is associated with several hepatobiliary diseases and a deleterious cancer, namely cholangiocarcinoma (Sripa et al., 2010). Despite intensive and continuous control programs and public health service activities having been introduced to control O. viverrini infection in Thailand (Jongsuksuntigul and Imsomboon, 2003), the prevalence in some areas, e.g. in Nakhon Phanom province, is still high (Sithithaworn et al., 2012). Health education aimed at stopping people from eating raw or undercooked fish is considered to be a key factor in FBT disease control (Jongsuksuntigul and Imsomboon, 2003); however culturally ingrained eating behaviors are difficult to change. Therefore, studying food safety procedures for health promotion may be an alternative way of controlling FBT disease. People in northeastern Thailand and Laos are likely to eat raw and semi-cooked fish regularly as part of their cuisines, in dishes such as koi-pla, lab-pla, som-khai pla, pla-som and pla-ra. In addition to koi-pla and lab-pla, which usually contain uncooked fish, pla-som is a very popular food product due to its availability for purchase at most local
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markets (Grundy-Warr et al., 2012). Although there is no direct evidence showing that eating pla-som can cause O. viverrini infection in humans, there is evidence suggesting that eating raw or uncooked fish such as pla-som, pla-ra and Koi-Pla, are correlated with O. viverrini infection, particularly in villagers in northeast Thailand (IARC, 1994; Sripa et al., 2011). Moreover, a recent study has revealed that contamination of FBT metacercariae including O. viverrini and Haplorchis taichui was found in pla-som being sold in markets in northeastern Thailand (Onsurathum et al., 2016). Thus, demonstrations on how to cook pla-som safely may aid in controlling FBT infection. Based on our previous observations, O. viverrini metacercariae (OVMC) were found in six species of fish, i.e. Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti and Puntioplites proctozysron (Pinlaor et al., 2013), all of these are usually used in pla-som preparation (Onsurathum et al., 2016). Thus, safe preparation methods are required to eliminate OVMC in pla-som. The present study attempted to investigate the effects of the duration of fermentation (the most important procedure for pla-som preparation) and low temperature on the viability and infectivity of OVMC in pla-som. In addition, infectivity of OVMC isolated from pla-som that had been prepared in several different ways was also tested by feeding it to hamsters. This study may be applied in the safe preparation of fermented fish to prevent O. viverrini infection.
5 days. At each of the desired time points, the refrigerated fish (approximately 0.40 kg) was taken from a refrigerator and mixed with 15 g of sticky rice, 21 g of minced garlic and 30 g of table salt. The Pla-som was put into plastic bags, tightly packed, and stored at room temperature for 3 days. Then, it was digested with artificial pepsin and OVMC was isolated, the viability of which was observed under a stereomicroscope. 2.4. OVMC isolation and identification
2. Materials and methods
OVMC were isolated by artificial digestion with the pepsin and sedimentation method, as has previously described elsewhere (Pinlaor et al., 2013). 0.33 or 0.40 kg of pla-som from each experimental groups 1 and 2, respectively, was mixed with 0.25% pepsin–1.5% HCl in 0.85% NaCl solution and then homogenized using a blender. Each mixture was incubated at 37 °C in a shaking water bath for 1 h. The digested materials were passed through sieves with different pore sizes (1000, 300, 106 and 250 μm) and then washed with 0.85% NaCl in a sedimentation jar until the supernatant became clear. Viable OVMC were determined by observation of the size of cysts under a stereomicroscope, as well as by the presence of a double-walled cyst, oval shape, clearly defined oral and ventral suckers, and display of vigorous movement within the cyst. Degeneration of metacercariae was determined by incomplete or destroyed structure, as previously described (Fan, 1998; Sukontason et al., 1998).
2.1. Fish collection and experiment design
2.5. Infectivity of OVMC to animal host
The study was conducted from August 2014 through January 2015. Cyprinid freshwater fish were obtained from endemic areas in Mukdahan province, northeastern Thailand, and in Khammouane province, Laos. These fish were placed in an icebox and transported to the laboratory. Wild fish species that could likely be contaminated with OVMC – including Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti and Puntioplites proctozystron (Pinlaor et al., 2013) – were selected and included in the study. At least eight kilograms of fish was used and randomly divided into two experimental groups. Fish in the first group was used to investigate the effect of pla-som fermentation time on the viability and infectivity of OVMC. Fish in the second experimental group was used to investigate the viability and infectivity of OVMC isolated from pla-som kept at a low temperature (4 °C). In addition, OVMC obtained from freshwater fish without fermentation was used as an uncooked control. All experiments were performed in duplicate.
Forty-four male golden Syrian hamsters weighing approximately 120 g were used. Hamsters were obtained from the Animal Unit, Faculty of Medicine, Khon Kaen University. Most of hamsters were infected orally with 50 OVMC in 0.85% NaCl solution (Tables 1 and 2) using the gastric intubation method. After 1 to 2 months post-infection, all hamsters were euthanized by diethyl ether inhalation and the liver of each animal was removed and examined for adult worms of O. viverrini. Those with positive findings were counted and recorded. The experimental protocol was approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU10/2558). The efficacy of OVMC to infect a host is evaluated by worm recovery (the total number of recovered worms divided by the number of OVMC infections), as described elsewhere (Fan, 1998).
2.2. Pla-som preparation and the effect of fermentation time on the viability of OVMC
The statistical significance of the degeneration of OVMC, worm recovery, storage time of refrigerated fish and fermentation time of pla-som were analyzed by the non-parametric Mann–Whitney U test. Pearson's correlation coefficient was used to analyze the correlation between pH and the viability of OVMC. Statistical analysis was performed using SPSS version 19 (SPSS Inc., Chicago, IL, USA). A P-value less than 0.05 was considered statistically significant.
Pla-som was prepared by a traditional method in both experiments. Approximately 2 kg of fish was first washed briefly in running tap water. After opening the body cavity of each fish and removing the intestines, the fish was mixed thoroughly with 75 g of glutinous rice, 105 g of minced garlic and 150 g of table salt. After mixing well, fish were divided equally into 7 groups (approximately 0.33 kg, each), put into separate plastic bags, tightly packed, and stored at room temperature for 1 to 7 days. After fermentation at the designated time point, each group of pla-som was taken for OVMC isolation and then the viability of metacercariae was observed under a stereomicroscope. The pH level of the pla-som was measured using a pH meter (500 series; Beckman Coulter, Brea, CA, USA). 2.3. Effect of low-temperature fish storage on viability of OVMC prior to pla-som processing A total of 2 kg of cyprinid fish possibly contaminated with OVMC was divided into five groups and kept in a refrigerator (4 °C) for 1 to
2.6. Data analysis
3. Results and discussion 3.1. Effect of fermentation time on viability and infectivity of OVMC Among northeastern and northern Thais, pla-ra and pla-som are often eaten daily (Grundy-Warr et al., 2012). This is a practice which, in addition to the consumption of koi-pla or lab-pla, might increase risk for FBT infection (Sukontason et al., 1998; Upatham et al., 1982). Data from interviews with villagers revealed that eating pla-ra was known to be a possible source of liver fluke infection (Jadsri and Noojoy, 1999). A recent experimental study showed that viable OVMC could not be identified in pla-ra fermented for 3 days or more (Prasongwatana et al., 2013). Typically, pla-ra is fermented for many
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Table 1 Effect of pla-som fermentation periods at room temperature on the infectivity of Opisthorchis viverrini metacercariae (OVMC). Groups
Uncooked control Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
pH (average)
NA 4.8 4.4 4.5 4.7 4.4 4.6 4.2
No. of administered OVMC/animal
50 50 50 40, 22 50 50 20 18
No. of hamsters
13 4 3 2 2 4 1 1
No. of infected hamsters (%)
13 (100) 4 (100) 3 (100) 1 (50) 1 (50) 0 0 0
Worm recovery No. of worm
%
P-Value
354 104 67 7 1 0 0 0
54.5 52 44.7 11.3 1 0 0 0
0.570 0.309 0.027* 0.027* 0.003* 0.027* 0.027*
NA = data not available, % of worm recovery was calculated by the total number of recovered worms divided by the number of infected OVMC. ⁎ : statistically significant (P b 0.05).
months before eating, while pla-som is usually fermented and eaten within one week. At day 3 of fermentation, viable OVMC were found in pla-som but not in pla-ra (Prasongwatana et al., 2013), implying that pla-som presents a higher risk of infection with OVMC as opposed to pla-ra. Based on this observation, it was of interest to test if the length of pla-som fermentation time would affect the ability of OVMC to infect the host. The present results showed that during a seven-day period of pla-som fermentation, viable OVMC were observed from day 1 to day 4 (Fig. 1A), while the percentage of degeneration of metacercariae increased rapidly after day 4 (Fig. 1B). The appearance of metacercariae was consistent with fermented fish morphology. The percentage of degenerated metacercariae increased from 0% (uncooked control) to 5.67%, 7.04%, 27.5%, 79% and 93.75% on days 1, 2, 3, 4 and 5, respectively, and reached 100% on days 6 and 7 of fermentation. The viable OVMC were confirmed by their infectivity to hamsters. The percentage of worm recovery for the uncooked control was 54.5% and then decreased to 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively, of pla-som fermentation (Table 1). A significant decrease in worm recovery was observed from day 3 to day 7 of fermentation time (P = 0.027, 0.027, 0.003, 0.027 and 0.027, respectively) when compared with the uncooked control group (Fig. 1B and 1C). Moreover, a negative correlation between pH and OVMC viability was found (R = − 0.314), suggesting that a reduction in pH during these periods may affect the viability and infectivity of OVMC. Although acidic conditions did not affect metacercariae excystation (Petney et al., 2013), it may cause metacercariae degeneration, resulting in an inability to infect the host during this period. Moreover, other factors such as high salt concentration may also affect metacercariae morphology and infectivity (Kruatrachue et al., 1982). Therefore, in order to avoid liver fluke infection, only raw pla-som that has been fermented for five days or more should be consumed.
3.2. Effect of low temperature on viability and infectivity of OVMC To mimic the production process of pla-som marketed for sale, in which fish are kept in cold conditions before fermentation, we refrigerated fish prior to processing in order to investigate the effect of low temperature on OVMC morphology and infectivity. People typically eat pla-som after fermentation for 3 days or more because the taste becomes more sour or acidic. In this experiment, viable OVMC, which were able to infect hamsters, were found in fish refrigerated for 1 or 2 days (Fig. 2A). The percentage of degenerated metacercariae increased from 0% (uncooked control) to 1.42%, 13.33% and 48.5% on days 1, 2 and 3, respectively. Completely degenerated metaceracriae were observed on days 4 and 5 of fermentation. The percentage of the worm recovery in hamsters was 54.5% (uncooked control), 3.3% and 12.7% for days 1 and 2, respectively (Table 2). A significant decrease in worm recovery was observed from day 1 to day 5 (P = 0.009, 0.010, 0.008, 0.008, and 0.008 for day 1 to day 5, respectively, compared to control). In addition, there was a negative correlation between pH and viability of OVMC (R = − 0.801), which was similar finding with the first experiment. After 3 days of refrigeration prior to pla-som preparation, about 50% of metacercariae were found to be degenerated (Fig. 2B) and were unable to infect the hamsters (Fig. 2C), indicating that low temperature might have affected infectivity of OVMC, in a similar manner as in other trematodes (Boray and Enigk, 1964; Ferrell et al., 2001). However, in a previous study, viable OVMC were observed even when kept in normal saline solution in a refrigerator for 1 month (Sithithaworn et al., 1991), suggesting that viability and infectivity of OVMC may also affected by other factors such as saline (Kruatrachue et al., 1982; Prasongwatana et al., 2013) and acidic conditions (Petney et al., 2013). However, in the case of metacercariae of C. sinensis, freezing or storing infected freshwater fish in heavy salt may not be effective in preventing infection of the host (Fan, 1998), suggesting that it may due to a difference in thickening of the cyst wall of C. sinensis metacercaria and OVMC
Table 2 Effect of fish storage periods at a refrigeration temperature before pla-som processing on the infectivity of Opisthorchis viverrini metacercariae (OVMC). Groups
Uncooked control Day 1 Day 2 Day 3 Day 4 Day 5
pH (average)
NA 4.8 4.4 4.6 4.3 4.1
No. of administered OVMC/animal
50 50 50, 50, 18 50 50 50, 32
No. of hamsters
13 3 3 3 3 2
No. of infected hamsters (%)
13 (100) 3 (100) 2 (66.7) 0 0 0
NA = data not available, % of worm recovery was calculated by the total number of recovered worms divided by the number of infected OVMC. ⁎ : statistically significant (P b 0.05).
Worm recovery No. of worm
%
p-value
354 5 15 0 0 0
54.5 3.3 12.7 0 0 0
0.009* 0.010* 0.008* 0.008* 0.008*
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Fig. 1. Morphology of pla-som and Opisthorchis viverrini metacercariae (OVMC) (A), percentage of degenerated OVMC (B) and worm recovery (C) after pla-som fermentation for seven days.
(Vajrasthira et al., 1961). Moreover, different ingredients and methods of cooking may affect OVMC viability. For instance, different fermented cyprinid fish dishes – including traditional pla-ra, lap-pla, and pla-
som – had varying effects on the viability of OVMC (Prasongwatana et al., 2013). Nevertheless, the study could not investigate the viability and infectivity of minute intestinal flukes such as Haplorchis spp.
Fig. 2. Morphology of pla-som and Opisthorchis viverrini metacercariae (OVMC) (A), percentage of degenerated OVMC (B) and worm recovery (C) after refrigerating fish for five days prior fermentation.
S. Onsurathum et al. / International Journal of Food Microbiology 218 (2016) 1–5
and Centrocestus spp. metacercariae in pla-som due to the fact that no contamination by these parasites was identified in fish. Further study on this is required. 4. Conclusions The present results demonstrate that the viability and infectivity of OVMC to the host differ depending on the pla-som fermentation period. Pla-som fermentation for 4 days or less increases the possibility of infection with OVMC. Thus, one important way of preventing O. viverrini infection would be to cook pla-som well before eating. Moreover, in order to prevent OVMC infection in those who consume pla-som, fish should be stored in a refrigerator for 3 days before the fermentation process begins. The results of this study may help to promote healthier eating habits among persons who often consume raw fish, and also provide health education for manufacturers and producers of fermented fish dishes. Acknowledgments This work was supported by the Higher Education Research Promotion and National Research Universities Project, Office of the Higher Education Commission, Thailand, through the Health Cluster, Khon Kaen University (SHeP-GMS, NRU542011) and The Royal Golden Jubilee Ph.D. Program (to SO and SP) (PHD/0167/2556). We thank the research assistants at the Faculty of Medicine, Khon Kaen University, for technical support. We also would like to acknowledge Prof. Christopher C. Salisbury and Mr. Dylan Southard for their editing the MS. References Boray, J.C., Enigk, K., 1964. Laboratory studies on the survival and infectivity of Fasciola hepatica- and F. Gigantica-Metacercariae. Z. Tropenmed. Parasitol. 15, 324–331. Chai, J.Y., Darwin, M.K., Lymbery, A.J., 2005. Fish-borne parasitic zoonoses: status and issues. Int. J. Parasitol. 35 (11–12), 1233–1254. Fan, P.C., 1998. Viability of metacercariae of Clonorchis sinensis in frozen or salted freshwater fish. Int. J. Parasitol. 28 (4), 603–605. Ferrell, D.L., Negovetich, N.J., Wetzel, E.J., 2001. Effect of temperature on the infectivity of metacercariae of Zygocotyle lunata (Digenea: Paramphistomidae). J. Parasitol. 87 (1), 10–13. Grundy-Warr, C., Andrews, R.H., Sithithaworn, P., Petney, T.N., Sripa, B., Laithavewat, L., Ziegler, A.D., 2012. Raw attitudes, wetland cultures, life-cycles: socio-cultural dynamics relating to Opisthorchis viverrini in the mekong basin. Parasitol. Int. 61 (1), 65–70. IARC, 1994. Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis). IARC monographs on the evaluation of carcinogenic risks to
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Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Effects of fermentation time and low temperature during the production process of Thai pickled fish (pla-som) on the viability and infectivity of Opisthorchis viverrini metacercariae Sudarat Onsurathum a,b, Porntip Pinlaor b,c, Ornuma Haonon a,b, Apisit Chaidee a,b, Lakhanawan Charoensuk a,b, Kitti Intuyod b,d, Thidarut Boonmars a,b, Porntip Laummaunwai a, Somchai Pinlaor a,b,⁎ a
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, Thailand c Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand d Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e
i n f o
Article history: Received 27 April 2015 Received in revised form 27 October 2015 Accepted 8 November 2015 Available online 10 November 2015 Keywords: Opisthorchis viverrini Metacercariae Freshwater cyprinid fish Pla-som fermentation Food safety
a b s t r a c t Contamination of a popular fermented fish dish, pla-som, by Opisthorchis viverrini metacercariae (OVMC) is a possible cause of carcinogenic liver fluke infection in Thailand. Affected individuals are at risk of bile duct cancer, which is a major health problem for people in the Greater Mekong Subregion. In order to investigate concerns about food safety, we studied the effects of fermentation time and low temperature on the viability and infectivity of OVMC during the pla-som production process. Pla-som was prepared at room temperature for up to 1 week in duplicate experiments using cyprinid freshwater fish obtained from an O. viverrini-endemic area. OVMC were then isolated and identified under a stereomicroscope. Complete and viable OVMC were found on days 1–4 of fermentation, while their morphology was degenerated thereafter. After OVMC were fed to hamsters, the percentage of the worm recovery after 1 to 2 months of infection was 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively. In order to measure the effect of low temperature on OVMC, fish were kept in a refrigerator (4 °C) for up to five days and then subsequently fermented for three days. In fish stored in a refrigerator for 1 and 2 days, viable OVMC were clearly observed and were able to infect hamsters, a worm-recovery percentage of 3.3% and 12.7%, respectively. By contrast, in pla-som prepared from fish stored for 3 to 5 days, OVMC were degenerated and could not infect the host. In conclusion, pla-som fermentation for more than four days and refrigerating fish for three days before pla-som processing can prevent O. viverrini infection. This study may increase awareness of fermented-fish dish preparation to prevent liver fluke infection. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Eating of semi-cooked or fermented fish dishes is associated with fish-borne trematode (FBT) infection because these foods usually contain metacercariae, an infective stage of trematode parasites, including minute intestinal and liver flukes (Chai et al., 2005; Keiser and Utzinger, 2009; Ziegler et al., 2013). Human small liver flukes consist of Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis. O. felineus is found in some parts of the European Union and the former Soviet Union, C. sinensis is found in eastern Asia, while O. viverrini is found in the Greater Mekong Subregion: Vietnam, Laos, Myanmar, Cambodia and Thailand (IARC, 2012). In Thailand, at least 6 million people are currently infected with the human liver fluke O. viverrini (Jongsuksuntigul and Imsomboon, 2003; Sithithaworn et al., 2012). ⁎ Corresponding author at: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (S. Pinlaor).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.11.001 0168-1605/© 2015 Elsevier B.V. All rights reserved.
Infection with O. viverrini is associated with several hepatobiliary diseases and a deleterious cancer, namely cholangiocarcinoma (Sripa et al., 2010). Despite intensive and continuous control programs and public health service activities having been introduced to control O. viverrini infection in Thailand (Jongsuksuntigul and Imsomboon, 2003), the prevalence in some areas, e.g. in Nakhon Phanom province, is still high (Sithithaworn et al., 2012). Health education aimed at stopping people from eating raw or undercooked fish is considered to be a key factor in FBT disease control (Jongsuksuntigul and Imsomboon, 2003); however culturally ingrained eating behaviors are difficult to change. Therefore, studying food safety procedures for health promotion may be an alternative way of controlling FBT disease. People in northeastern Thailand and Laos are likely to eat raw and semi-cooked fish regularly as part of their cuisines, in dishes such as koi-pla, lab-pla, som-khai pla, pla-som and pla-ra. In addition to koi-pla and lab-pla, which usually contain uncooked fish, pla-som is a very popular food product due to its availability for purchase at most local
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markets (Grundy-Warr et al., 2012). Although there is no direct evidence showing that eating pla-som can cause O. viverrini infection in humans, there is evidence suggesting that eating raw or uncooked fish such as pla-som, pla-ra and Koi-Pla, are correlated with O. viverrini infection, particularly in villagers in northeast Thailand (IARC, 1994; Sripa et al., 2011). Moreover, a recent study has revealed that contamination of FBT metacercariae including O. viverrini and Haplorchis taichui was found in pla-som being sold in markets in northeastern Thailand (Onsurathum et al., 2016). Thus, demonstrations on how to cook pla-som safely may aid in controlling FBT infection. Based on our previous observations, O. viverrini metacercariae (OVMC) were found in six species of fish, i.e. Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti and Puntioplites proctozysron (Pinlaor et al., 2013), all of these are usually used in pla-som preparation (Onsurathum et al., 2016). Thus, safe preparation methods are required to eliminate OVMC in pla-som. The present study attempted to investigate the effects of the duration of fermentation (the most important procedure for pla-som preparation) and low temperature on the viability and infectivity of OVMC in pla-som. In addition, infectivity of OVMC isolated from pla-som that had been prepared in several different ways was also tested by feeding it to hamsters. This study may be applied in the safe preparation of fermented fish to prevent O. viverrini infection.
5 days. At each of the desired time points, the refrigerated fish (approximately 0.40 kg) was taken from a refrigerator and mixed with 15 g of sticky rice, 21 g of minced garlic and 30 g of table salt. The Pla-som was put into plastic bags, tightly packed, and stored at room temperature for 3 days. Then, it was digested with artificial pepsin and OVMC was isolated, the viability of which was observed under a stereomicroscope. 2.4. OVMC isolation and identification
2. Materials and methods
OVMC were isolated by artificial digestion with the pepsin and sedimentation method, as has previously described elsewhere (Pinlaor et al., 2013). 0.33 or 0.40 kg of pla-som from each experimental groups 1 and 2, respectively, was mixed with 0.25% pepsin–1.5% HCl in 0.85% NaCl solution and then homogenized using a blender. Each mixture was incubated at 37 °C in a shaking water bath for 1 h. The digested materials were passed through sieves with different pore sizes (1000, 300, 106 and 250 μm) and then washed with 0.85% NaCl in a sedimentation jar until the supernatant became clear. Viable OVMC were determined by observation of the size of cysts under a stereomicroscope, as well as by the presence of a double-walled cyst, oval shape, clearly defined oral and ventral suckers, and display of vigorous movement within the cyst. Degeneration of metacercariae was determined by incomplete or destroyed structure, as previously described (Fan, 1998; Sukontason et al., 1998).
2.1. Fish collection and experiment design
2.5. Infectivity of OVMC to animal host
The study was conducted from August 2014 through January 2015. Cyprinid freshwater fish were obtained from endemic areas in Mukdahan province, northeastern Thailand, and in Khammouane province, Laos. These fish were placed in an icebox and transported to the laboratory. Wild fish species that could likely be contaminated with OVMC – including Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti and Puntioplites proctozystron (Pinlaor et al., 2013) – were selected and included in the study. At least eight kilograms of fish was used and randomly divided into two experimental groups. Fish in the first group was used to investigate the effect of pla-som fermentation time on the viability and infectivity of OVMC. Fish in the second experimental group was used to investigate the viability and infectivity of OVMC isolated from pla-som kept at a low temperature (4 °C). In addition, OVMC obtained from freshwater fish without fermentation was used as an uncooked control. All experiments were performed in duplicate.
Forty-four male golden Syrian hamsters weighing approximately 120 g were used. Hamsters were obtained from the Animal Unit, Faculty of Medicine, Khon Kaen University. Most of hamsters were infected orally with 50 OVMC in 0.85% NaCl solution (Tables 1 and 2) using the gastric intubation method. After 1 to 2 months post-infection, all hamsters were euthanized by diethyl ether inhalation and the liver of each animal was removed and examined for adult worms of O. viverrini. Those with positive findings were counted and recorded. The experimental protocol was approved by the Animal Ethics Committee of Khon Kaen University, Thailand (AEKKU10/2558). The efficacy of OVMC to infect a host is evaluated by worm recovery (the total number of recovered worms divided by the number of OVMC infections), as described elsewhere (Fan, 1998).
2.2. Pla-som preparation and the effect of fermentation time on the viability of OVMC
The statistical significance of the degeneration of OVMC, worm recovery, storage time of refrigerated fish and fermentation time of pla-som were analyzed by the non-parametric Mann–Whitney U test. Pearson's correlation coefficient was used to analyze the correlation between pH and the viability of OVMC. Statistical analysis was performed using SPSS version 19 (SPSS Inc., Chicago, IL, USA). A P-value less than 0.05 was considered statistically significant.
Pla-som was prepared by a traditional method in both experiments. Approximately 2 kg of fish was first washed briefly in running tap water. After opening the body cavity of each fish and removing the intestines, the fish was mixed thoroughly with 75 g of glutinous rice, 105 g of minced garlic and 150 g of table salt. After mixing well, fish were divided equally into 7 groups (approximately 0.33 kg, each), put into separate plastic bags, tightly packed, and stored at room temperature for 1 to 7 days. After fermentation at the designated time point, each group of pla-som was taken for OVMC isolation and then the viability of metacercariae was observed under a stereomicroscope. The pH level of the pla-som was measured using a pH meter (500 series; Beckman Coulter, Brea, CA, USA). 2.3. Effect of low-temperature fish storage on viability of OVMC prior to pla-som processing A total of 2 kg of cyprinid fish possibly contaminated with OVMC was divided into five groups and kept in a refrigerator (4 °C) for 1 to
2.6. Data analysis
3. Results and discussion 3.1. Effect of fermentation time on viability and infectivity of OVMC Among northeastern and northern Thais, pla-ra and pla-som are often eaten daily (Grundy-Warr et al., 2012). This is a practice which, in addition to the consumption of koi-pla or lab-pla, might increase risk for FBT infection (Sukontason et al., 1998; Upatham et al., 1982). Data from interviews with villagers revealed that eating pla-ra was known to be a possible source of liver fluke infection (Jadsri and Noojoy, 1999). A recent experimental study showed that viable OVMC could not be identified in pla-ra fermented for 3 days or more (Prasongwatana et al., 2013). Typically, pla-ra is fermented for many
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Table 1 Effect of pla-som fermentation periods at room temperature on the infectivity of Opisthorchis viverrini metacercariae (OVMC). Groups
Uncooked control Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
pH (average)
NA 4.8 4.4 4.5 4.7 4.4 4.6 4.2
No. of administered OVMC/animal
50 50 50 40, 22 50 50 20 18
No. of hamsters
13 4 3 2 2 4 1 1
No. of infected hamsters (%)
13 (100) 4 (100) 3 (100) 1 (50) 1 (50) 0 0 0
Worm recovery No. of worm
%
P-Value
354 104 67 7 1 0 0 0
54.5 52 44.7 11.3 1 0 0 0
0.570 0.309 0.027* 0.027* 0.003* 0.027* 0.027*
NA = data not available, % of worm recovery was calculated by the total number of recovered worms divided by the number of infected OVMC. ⁎ : statistically significant (P b 0.05).
months before eating, while pla-som is usually fermented and eaten within one week. At day 3 of fermentation, viable OVMC were found in pla-som but not in pla-ra (Prasongwatana et al., 2013), implying that pla-som presents a higher risk of infection with OVMC as opposed to pla-ra. Based on this observation, it was of interest to test if the length of pla-som fermentation time would affect the ability of OVMC to infect the host. The present results showed that during a seven-day period of pla-som fermentation, viable OVMC were observed from day 1 to day 4 (Fig. 1A), while the percentage of degeneration of metacercariae increased rapidly after day 4 (Fig. 1B). The appearance of metacercariae was consistent with fermented fish morphology. The percentage of degenerated metacercariae increased from 0% (uncooked control) to 5.67%, 7.04%, 27.5%, 79% and 93.75% on days 1, 2, 3, 4 and 5, respectively, and reached 100% on days 6 and 7 of fermentation. The viable OVMC were confirmed by their infectivity to hamsters. The percentage of worm recovery for the uncooked control was 54.5% and then decreased to 52%, 44.7%, 11.3% and 1% for days 1, 2, 3 and 4, respectively, of pla-som fermentation (Table 1). A significant decrease in worm recovery was observed from day 3 to day 7 of fermentation time (P = 0.027, 0.027, 0.003, 0.027 and 0.027, respectively) when compared with the uncooked control group (Fig. 1B and 1C). Moreover, a negative correlation between pH and OVMC viability was found (R = − 0.314), suggesting that a reduction in pH during these periods may affect the viability and infectivity of OVMC. Although acidic conditions did not affect metacercariae excystation (Petney et al., 2013), it may cause metacercariae degeneration, resulting in an inability to infect the host during this period. Moreover, other factors such as high salt concentration may also affect metacercariae morphology and infectivity (Kruatrachue et al., 1982). Therefore, in order to avoid liver fluke infection, only raw pla-som that has been fermented for five days or more should be consumed.
3.2. Effect of low temperature on viability and infectivity of OVMC To mimic the production process of pla-som marketed for sale, in which fish are kept in cold conditions before fermentation, we refrigerated fish prior to processing in order to investigate the effect of low temperature on OVMC morphology and infectivity. People typically eat pla-som after fermentation for 3 days or more because the taste becomes more sour or acidic. In this experiment, viable OVMC, which were able to infect hamsters, were found in fish refrigerated for 1 or 2 days (Fig. 2A). The percentage of degenerated metacercariae increased from 0% (uncooked control) to 1.42%, 13.33% and 48.5% on days 1, 2 and 3, respectively. Completely degenerated metaceracriae were observed on days 4 and 5 of fermentation. The percentage of the worm recovery in hamsters was 54.5% (uncooked control), 3.3% and 12.7% for days 1 and 2, respectively (Table 2). A significant decrease in worm recovery was observed from day 1 to day 5 (P = 0.009, 0.010, 0.008, 0.008, and 0.008 for day 1 to day 5, respectively, compared to control). In addition, there was a negative correlation between pH and viability of OVMC (R = − 0.801), which was similar finding with the first experiment. After 3 days of refrigeration prior to pla-som preparation, about 50% of metacercariae were found to be degenerated (Fig. 2B) and were unable to infect the hamsters (Fig. 2C), indicating that low temperature might have affected infectivity of OVMC, in a similar manner as in other trematodes (Boray and Enigk, 1964; Ferrell et al., 2001). However, in a previous study, viable OVMC were observed even when kept in normal saline solution in a refrigerator for 1 month (Sithithaworn et al., 1991), suggesting that viability and infectivity of OVMC may also affected by other factors such as saline (Kruatrachue et al., 1982; Prasongwatana et al., 2013) and acidic conditions (Petney et al., 2013). However, in the case of metacercariae of C. sinensis, freezing or storing infected freshwater fish in heavy salt may not be effective in preventing infection of the host (Fan, 1998), suggesting that it may due to a difference in thickening of the cyst wall of C. sinensis metacercaria and OVMC
Table 2 Effect of fish storage periods at a refrigeration temperature before pla-som processing on the infectivity of Opisthorchis viverrini metacercariae (OVMC). Groups
Uncooked control Day 1 Day 2 Day 3 Day 4 Day 5
pH (average)
NA 4.8 4.4 4.6 4.3 4.1
No. of administered OVMC/animal
50 50 50, 50, 18 50 50 50, 32
No. of hamsters
13 3 3 3 3 2
No. of infected hamsters (%)
13 (100) 3 (100) 2 (66.7) 0 0 0
NA = data not available, % of worm recovery was calculated by the total number of recovered worms divided by the number of infected OVMC. ⁎ : statistically significant (P b 0.05).
Worm recovery No. of worm
%
p-value
354 5 15 0 0 0
54.5 3.3 12.7 0 0 0
0.009* 0.010* 0.008* 0.008* 0.008*
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Fig. 1. Morphology of pla-som and Opisthorchis viverrini metacercariae (OVMC) (A), percentage of degenerated OVMC (B) and worm recovery (C) after pla-som fermentation for seven days.
(Vajrasthira et al., 1961). Moreover, different ingredients and methods of cooking may affect OVMC viability. For instance, different fermented cyprinid fish dishes – including traditional pla-ra, lap-pla, and pla-
som – had varying effects on the viability of OVMC (Prasongwatana et al., 2013). Nevertheless, the study could not investigate the viability and infectivity of minute intestinal flukes such as Haplorchis spp.
Fig. 2. Morphology of pla-som and Opisthorchis viverrini metacercariae (OVMC) (A), percentage of degenerated OVMC (B) and worm recovery (C) after refrigerating fish for five days prior fermentation.
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and Centrocestus spp. metacercariae in pla-som due to the fact that no contamination by these parasites was identified in fish. Further study on this is required. 4. Conclusions The present results demonstrate that the viability and infectivity of OVMC to the host differ depending on the pla-som fermentation period. Pla-som fermentation for 4 days or less increases the possibility of infection with OVMC. Thus, one important way of preventing O. viverrini infection would be to cook pla-som well before eating. Moreover, in order to prevent OVMC infection in those who consume pla-som, fish should be stored in a refrigerator for 3 days before the fermentation process begins. The results of this study may help to promote healthier eating habits among persons who often consume raw fish, and also provide health education for manufacturers and producers of fermented fish dishes. Acknowledgments This work was supported by the Higher Education Research Promotion and National Research Universities Project, Office of the Higher Education Commission, Thailand, through the Health Cluster, Khon Kaen University (SHeP-GMS, NRU542011) and The Royal Golden Jubilee Ph.D. Program (to SO and SP) (PHD/0167/2556). We thank the research assistants at the Faculty of Medicine, Khon Kaen University, for technical support. We also would like to acknowledge Prof. Christopher C. Salisbury and Mr. Dylan Southard for their editing the MS. References Boray, J.C., Enigk, K., 1964. Laboratory studies on the survival and infectivity of Fasciola hepatica- and F. Gigantica-Metacercariae. Z. Tropenmed. Parasitol. 15, 324–331. Chai, J.Y., Darwin, M.K., Lymbery, A.J., 2005. Fish-borne parasitic zoonoses: status and issues. Int. J. Parasitol. 35 (11–12), 1233–1254. Fan, P.C., 1998. Viability of metacercariae of Clonorchis sinensis in frozen or salted freshwater fish. Int. J. Parasitol. 28 (4), 603–605. Ferrell, D.L., Negovetich, N.J., Wetzel, E.J., 2001. Effect of temperature on the infectivity of metacercariae of Zygocotyle lunata (Digenea: Paramphistomidae). J. Parasitol. 87 (1), 10–13. Grundy-Warr, C., Andrews, R.H., Sithithaworn, P., Petney, T.N., Sripa, B., Laithavewat, L., Ziegler, A.D., 2012. Raw attitudes, wetland cultures, life-cycles: socio-cultural dynamics relating to Opisthorchis viverrini in the mekong basin. Parasitol. Int. 61 (1), 65–70. IARC, 1994. Infection with liver flukes (Opisthorchis viverrini, Opisthorchis felineus and Clonorchis sinensis). IARC monographs on the evaluation of carcinogenic risks to
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