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Effects of high pressure processing on hatching of eggs of the zoonotic rat tapeworm Hymenolepis diminuta
Veterinary Parasitology 176 (2011) 185–188
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Effects of high pressure processing on hatching of eggs of the zoonotic rat tapeworm Hymenolepis diminuta A.M.A. Merwad a , S.M. Mitchell b , A.M. Zajac c , G.J. Flick d , D.S. Lindsay c,∗ a b c d
Department of Zoonosis Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt SCYNEXIS, Inc. P.O. Box 12878, Research Triangle Park, NC 27709, USA Department of Biomedical Science and Pathology, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061, USA Department of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 14 September 2010 Received in revised form 23 October 2010 Accepted 26 October 2010 Keywords: Hymenolepis diminuta Rat tapeworm High hydrostatic pressure Contamination Inactivation
a b s t r a c t High hydrostatic pressure processing (HPP) is an effective non-thermal treatment to remove pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms but has had limited study on eukaryotic organisms. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and Ascaris suum eggs. Nothing is known on the efficacy of HPP on tapeworm cysts or eggs. Eggs of important zoonotic tapeworms including Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption. The present study examined the efficacy of HPP on the viability of Hymenolepis diminuta eggs. Efficacy of HPP treatment was measured using an egg hatch assay in two experiments. One thousand unhatched H. diminuta eggs in Hanks balanced salt solution were packaged in sealable bags and exposed to 100–600 megapascals (MPa; 1 MPa = 10 atm = 147 psi) for 60 s in a commercial HPP unit. Positive (no HPP) and negative (No HPP but frozen/thawed) controls were examined in each experiment. None of the HPP untreated and frozen eggs (negative controls) were able to hatch or exclude trypan blue when placed in the hatching solution in experiment 1 or 2. HPP untreated and nonfrozen eggs (positive controls) hatched and excluded trypan blue; 75% were positive in experiment 1 and 80% were positive in experiment 2. No hatched eggs were observed when they were exposed to 300–600 MPa for 60 s. Treatment at 400 MPa and above caused rupturing of the oncosphere. Results from this study indicate that HPP is a possible method to inactivate tapeworm eggs and that the susceptibility of tapeworm eggs to HPP is similar to or greater than that of nematode eggs or tissue larvae. © 2010 Elsevier B.V. All rights reserved.
1. Introduction. High pressure processing (HPP) is an effective nonthermal means of eliminating nonspore-forming bacteria from a variety of food products (Flick, 2003). The shelf life of the products is extended and the sensory features (appearance, texture, taste, smell, etc.) of the food are not, or are only minimally, affected by HPP. Other advantages of HPP
∗ Corresponding author. Tel.: +1 540 231 6302. fax: +1 540 231 3426. E-mail address: [email protected] (D.S. Lindsay). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.055
over traditional thermal processing include reduced processing times; minimal heat damage problems; retention of freshness, flavor, texture, color and no vitamin C loss (Tewari et al., 1999). Little has been published on the effects of HPP on zoonotic metazoan parasites. Ohnishi et al. (1992, 1994) determined that pressures of greater than 200 megapascals (MPa; 1 MPa = 10 atm = 147 psi) kill 8-wk-old Trichinella spiralis larvae. Gamble et al. (1998) determined 55–60 MPa did not kill all T. spiralis larvae in pork tenderloin or diaphragm. Treatment at 200 MPa for 10 min at temperatures between 0 and 15 ◦ C kills Anisakis simplex larvae with
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Table 1 Effects of high hydrostatic pressure (HPP) on hatching of eggs of Hymenolepis diminuta exposed to no or 100–600 megapascals (MPa) pressure for 60 s. Pressure
% Hatched experiment 1
% Hatched experiment 2
Mean
0 MPa, unfrozen 0 MPa, frozen 100 MPa 200 MPa 300 MPa 400 MPa 500 MPa 600 MPa
75 0 18 16 0 0 0 0
80 0 19 15 0 0 0 0
77.5 0 18.5 15.0 0 0 0 0
lack of motility used as an indicator of larval death (MolinaGarcia and Sanz, 2002). Dong et al. (2003) found the times and pressures required to kill 100% of A. simplex larvae in king salmon and arrowtooth flounder were 30–60 s at 414 MPa, 90–180 s at 276 MPa and 180 s at 207 MPa. Rosypal et al. (2007) have demonstrated that unembryonated Ascaris suum eggs fail to develop after exposure to pressures of 241 MPa or more for 60 s, or when exposed to 276 MPa for 10–30 s. Hymenolepis diminuta is a rat tapeworm parasite with beetle intermediate hosts. It is commonly found in beetles in areas used for grain storage or storage of other dry foods. Humans, usually children are accidental hosts of H. diminuta and become infected through ingestion of cysticercoids in infected beetles. H. diminuta infection has sporadically been reported in humans from developed nations (Edelman et al., 1965; Levi et al., 1987; Hamrick et al., 1990) but is more frequent in developing nations (Jones, 1979; Weisse and Raszka, 1996; Wiwanitkit, 2004; Patamia et al., 2010). Eggs of the important zoonotic tapeworms Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption (Dorny et al., 2009; Eckert and Deplazes, 2004). Nothing is known about the susceptibility of tapeworm eggs to HPP treatment. Therefore, the present study was conducted using H. diminuta eggs collected from experimentally infected rats. 2. Materials and methods 2.1. Hymenolepis diminuta eggs H. diminuta eggs were collected from the feces of experimentally infected white rats, Rattus norvegicus and concentrated by flotation in Sheather’s sugar solution. Isolated eggs were stored in Hanks balanced salt solution (HBSS) at 4 ◦ C until used. Prior to use, 1 × 103 eggs in 1 ml HBSS were placed in sealable bags (Kapak Sealpak pouches, Minneapolis, Minnesota, USA) and the bags compressed to force out air. The bags were then sealed with a sealing machine (Shop sealer model FS-315, Fugi Impulse Vietnam Co. Ltd., Sai Gon-Linh Trung EPZ, Japan). The bags were placed in additional sealable bags and sealed. This additional bag was used to prevent any potential contamination of the HPP unit. Control eggs were prepared in bags but were not subjected to HPP. The positive control egg containing bags were kept at 4 ◦ C in refrigerator until used for hatching experiments. Bags containing H. diminuta eggs to be killed
and used as negative controls were subjected to several freeze–thaw cycles by freezing in a liquid nitrogen tank and thawing at room temperature. These negative control (dead) eggs were kept at 4 ◦ C in refrigerator until used for hatching experiments. 2.2. High pressure processing The sealed bags containing H. diminuta eggs were placed into a commercial HPP unit (Quintus Food Press QFP 35L-600 Model, Flow International Corporation [Avure Technologies], Kent, Washington, USA) with 7XS-6000intensifier pump and maximum operating pressure of 600 MPa. The HPP unit was installed and operated at Virginia Tech’s Department of Food Science and Technology. All samples were exposed for 60 s. Pressures of 100, 200, 300, 400, 500, and 600 MPa were examined and two experiments were conducted (Table 1). 2.3. Egg hatching assay The egg-hatching assay described by Allen et al. (1965) was used. Briefly, after HPP treatment, the processed H. diminuta egg solution was aspirated into 15 ml conical centrifuge tubes and 0.5 ml Tyrode’s solution containing 2 mm glass beads (N = 20) was added to each tube. The tubes were vortexed for 15–30 s to disrupt or remove the egg-shell. The tubes were centrifuged for 10 min and the supernatant and glass beads were removed. The sediment in each centrifuged tube was placed in a new 15 ml conical centrifuge tube and 14 ml of hatching solution (50 mg ␣-amylase, 0.3 mg trypsin in 40 ml Tyrode’s solution) was added and the sample vortexed 15–30 s. The solution was incubated at room temperature for 30 min. The tubes were centrifuged and the sediment was resuspended in 2 ml of a mixture of trypan blue in HBSS (1.8 ml HBSS + 0.2 ml 0.4% trypan blue solution) to determine viability of the hatched oncospheres. Oncospheres that stained blue were considered nonviable. Examinations were done using an Olympus BH60 microscope equipped with differential contrast optics (Olympus America Inc., Center Valley, Pennsylvania, USA). A total of 100 HPP treated or controls eggs were counted in 100 l aliquots and categorized as viable or nonviable for each treatment. 3. Results H. diminuta eggs not frozen and not treated with HPP and exposed to hatching medium were viable and the hex-
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Fig. 1. Positive control Hymenolepis diminuta egg, not frozen and not treated with high hydrostatic pressure, hatching from egg placed in hatching medium. The hexacanth embryo (H) has exited through breaks in the inner (white arrowhead) and outer layers (arrow) of the eggshell. Cytoplasm (C) surrounds the hexacanth embryo and hooks (black arrowhead) are visible. Bar = 10 microns.
acanth embryo exited the eggs via a break in the eggshell (Fig. 1). Freezing in liquid nitrogen rendered 100% of the negative control eggs and various percentages of eggs treated at 100 or 200 MPa nonviable as judged by trypan blue exclusion (Fig. 2). No viable H. diminuta eggs were seen in samples treated with 300, 400, 500, or 600 MPa (Table 1). Treatment with these pressures induced breakage of the onchosphere wall and leakage of its contents in to the medium (Fig. 3). This was not observed in negative control (frozen) eggs.
Fig. 2. Nonviable negative control Hymenolepis diminuta treated with 200 MPa. The cytoplasm has taken up the trypan blue. Note the hooks arrows (arrows). Bar = 10 microns.
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Fig. 3. Hymenolepis diminuta egg treated with 500 MPa high hydrostatic pressure and demonstrating abnormal appearance. The covering (arrowhead) of the embryo has been disrupted and contents are leaking (arrow) into hatching medium. One pair of hooks (white arrowhead) has moved through an apparent disruption in the parasite covering membrane while the other two pairs of hooks remain inside the covering membrane (white arrows). Bar = 10 microns.
4. Discussion The effects of HPP treatment on the viability and structure of several protozoan parasites have been examined. Cryptosporidium parvum oocysts (Slifko et al., 2000; Collins et al., 2005), Encephalitozoon cuniculi spores (Jordan et al., 2005), Toxoplasma gondii oocysts (Lindsay et al., 2005, 2008) and tissue cysts (Lindsay et al. 2006), and Eimeria acervulina oocysts (Kniel et al., 2007) have been tested for the efficacy of HPP to inactivate the infectivity of these protozoans. Slifko et al. (2000) examined the effects of 550 MPa on C. parvum oocysts in apple and orange juice. They determined that a 60 s exposure at 550 MPa was 100% effective in decreasing infectivity of oocysts for cell cultures. The effects of HPP on the infectivity of C. parvum oocysts recovered from experimentally exposed HPP treated oysters for neonatal mice were examined by Collins et al. (2005). They found that a 180 s exposure to 550 MPa produced the maximum decrease in numbers of C. parvum positive mouse pups (93%) (Collins et al., 2005). Neither study (Slifko et al., 2000; Collins et al., 2005) examined the structural effects induced by HPP treatment on C. parvum oocysts. Jordan et al. (2005) examined the effects of HPP treatment of E. cuniculi spores on subsequent development in vitro. Spores treated with 200 to 275 MPa for 60 s had reduced infectivity for host cells. After treatment of 345 MPa or more for 60 s, spores were unable to infect host cells (Jordan et al., 2005). No structural alterations were observed in spores when they were examined using transmission electron microscopy after treatment with up to 480 MPa for 60 s min (Jordan et al., 2005). T. gondii oocysts in HBSS, dis-
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tilled water or spot-inoculated onto raspberries and treated with HPP for 60 s at ≥340 MPa were rendered noninfectious for mice (Lindsay et al., 2005, 2008). No structural alterations occurred in these oocysts using light microscopy. Tissue cysts of T. gondii in ground pork were rendered noninfectious for mice by treatment with ≥300 MPa for 30–90 s (Lindsay et al., 2006). They were not examined for structural alterations. Oocysts of E. acervulina collected from experimentally contaminated raspberries and basil treated with 550 MPa at 40 ◦ C for 2 min were rendered noninfectious for 3-wk-old chickens (Kniel et al., 2007). Adverse affects on the oocyst wall were not a cause for the loss of infectivity because the oocysts retained the ability to exclude methylene blue (Kniel et al., 2007) Rosypal et al. (2007) reported that no morphological alterations were noted with light microscopy when unembryonated A. suum eggs treated with 552 MPa for 60 s were compared to untreated controls. Ohnishi et al. (1994) examined the histochemical and morphological effects of HPP (10 min at 25 ◦ C) on T. spiralis in muscle tissues from experimentally infected mice after the tissue had been paraffin embedded and sections processed using Azan, periodic acid-Schiff (PAS), and routine hematoxylin and eosin (H&E) staining. At pressures of ≤100 MPa T. spiralis larvae stained normally. Alteration in larval structure associated with decreased Azan staining of stichocytes was observed at ≥150 MPa. They attributed this to acidiophilic structures becoming basophilic after HPP (Ohnishi et al., 1994). They also note decreased distortion of PAS positive staining of stichocytes treated with 300 MPa. They suggested that glycogen and/or glycoprotein’s demonstrated by PAS staining may decompose after HPP (Ohnishi et al., 1994). Acknowledgements The contribution of AMAM was supported by Partner and Ownership grant belonging to Ministry of Higher Education and Scientific Research, Egypt. This study was financially supported in part by an Animal Health and Disease grant to DSL and GJF. We thank Laura S. Douglas for her help with HPP treatment operations. References Allen, K.I., Bentzen, I., Voge, M., 1965. In vitro hatching of onchospheres of four hymenolepid cestodes. J. Parasitol. 51, 235–242. Collins, M.V., Flick, G.J., Smith, S.A., Fayer, R., Croonenberghs, R., O’Keefe, S., Lindsay, D.S., 2005. The effect of high-pressure processing on infectivity of Cryptosporidium parvum oocysts recovered from experimentally exposed Eastern oysters (Crassostrea virginica). J. Eukaryot. Microbiol. 52, 500–504.
Dong, F.M., Cook, A.R., Herwig, R.P., 2003. High hydrostatic pressure treatment of finfish to inactivate Anisakis simplex. J. Food Prot. 66, 1924–1926. Dorny, P., Praet, N., Deckers, N., Gabriel, S., 2009. Emerging food-borne parasites. Vet. Parasitol. 163, 196–206. Eckert, J., Deplazes, P., 2004. Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern. Clin. Microbiol. Rev. 17, 107–135. Edelman, M.H., Spingarn, C.L., Nauenverg, W.G.C., Gregory, C., 1965. Hymenolepis diminuta (rat tapeworm) infection in man. Am. J. Med. 38, 951–953. Flick Jr., G.J., 2003. High pressure processing offers varied applications. Global Aquacult. Adv. 6, 82–85. Gamble, H.R., Solomon, M.B., Long, J.B., 1998. Effects of hydrodynamic pressure on the viability of Trichinella spiralis in pork. J. Food Prot. 61, 637–639. Hamrick, H.J., Bowdre, J.H., Church, S.M., 1990. Rat tapeworm (Hymenolepis diminuta) infection in a child. Pediatr. Infect. Dis. J. 9, 216–219. Jones, W.E., 1979. Niclosamide as a treatment for Hymenolepis diminuta and Dipylidium caninum infection in man. Am. J. Trop. Med. Hyg. 28, 300–302. Jordan, C.N., Zajac, A.M., Holliman, D., Flick, G.F., Lindsay, D.S., 2005. Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi. J. Parasitol. 91, 1487–1488. Kniel, K.E., Shearer, A.E.H., Cascarino, J.L., Wilkins, G.C., Jenkins, M.C., 2007. High hydrostatic pressure and UV light treatment of produce contaminated with Eimeria acervulina as a Cyclospora cayetanensis surrogate. J. Food Prot. 70, 2837–2842. Levi, M.H., Raucher, B.G., Teicher, E., Sheehan, D.J., McKitrick, J.C., 1987. Hymenolepis diminuta: one of three enteric pathogens isolated from a child. Diagn. Microbiol. Infect. Dis. 7, 255–259. Lindsay, D.S., Collins, M.V., Jordan, C.N., Flick, G.J., Dubey, J.P., 2005. Effects of high pressure processing on infectivity of Toxoplasma gondii oocysts for mice. J. Parasitol. 91, 699–701. Lindsay, D.S., Collins, M.V., Holliman, D., Flick, G.J., Dubey, J.P., 2006. Effects of high-pressure processing on Toxoplasma gondii tissue cysts in ground pork. J. Parasitol. 92, 195–196. Lindsay, D.S., Holliman, D., Flick, G.J., Goodwin, D.G., Mitchell, S.M., Dubey, J.P., 2008. Effects of high pressure processing on Toxoplasma gondii oocysts on raspberries. J. Parasitol. 94, 757–758. Molina-Garcia, A.D., Sanz, P.D., 2002. Anisakis simplex larva killed by high- hydrostatic-pressure processing. J. Food Prot. 65, 383– 388. Ohnishi, Y., Ono, T., Shigehisa, T., Ohmori, T., 1992. Effect of high hydrostatic pressure on muscle larvae of Trichinella spiralis. Jpn. J. Parasitol. 41, 373–377. Ohnishi, Y., Ono, T., Shigehisa, T., 1994. Histochemical and morphological studies on Trichinella spiralis larvae treated with high hydrostatic pressure. Int. J. Parasitol. 24, 425–427. Patamia, I., Cappello, E., Castellano-Chiodo, D., Greco, F., Nigro, L., Cacopardo, B., 2010. A human case of Hymenolepis diminuta in a child from eastern Sicily. Korean J. Parasitol. 48, 167–169. Rosypal, A.C., Bowman, D.D., Holliman, D., Flick, G.J., Lindsay, D.S., 2007. Effects of high hydrostatic pressure on embryonation of Ascaris suum eggs. Vet. Parasitol. 145, 86–89. Slifko, T.R., Raghubeer, E., Rose, J.B., 2000. Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity. J. Food Prot. 63, 1262–1267. Tewari, G., Jayas, D.S., Holley, A.L., 1999. High pressure processing of foods: An overview. Sci. Des. Aliments 19, 619–661. Weisse, M.E., Raszka Jr., W.V., 1996. Cestode infection in children. Adv. Pediatr. Infect. Dis. 12, 109–153. Wiwanitkit, V., 2004. Overview of Hymenolepis diminuta infection among Thai patients. MedGenMed 6, 7.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Effects of high pressure processing on hatching of eggs of the zoonotic rat tapeworm Hymenolepis diminuta A.M.A. Merwad a , S.M. Mitchell b , A.M. Zajac c , G.J. Flick d , D.S. Lindsay c,∗ a b c d
Department of Zoonosis Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt SCYNEXIS, Inc. P.O. Box 12878, Research Triangle Park, NC 27709, USA Department of Biomedical Science and Pathology, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061, USA Department of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 14 September 2010 Received in revised form 23 October 2010 Accepted 26 October 2010 Keywords: Hymenolepis diminuta Rat tapeworm High hydrostatic pressure Contamination Inactivation
a b s t r a c t High hydrostatic pressure processing (HPP) is an effective non-thermal treatment to remove pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms but has had limited study on eukaryotic organisms. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and Ascaris suum eggs. Nothing is known on the efficacy of HPP on tapeworm cysts or eggs. Eggs of important zoonotic tapeworms including Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption. The present study examined the efficacy of HPP on the viability of Hymenolepis diminuta eggs. Efficacy of HPP treatment was measured using an egg hatch assay in two experiments. One thousand unhatched H. diminuta eggs in Hanks balanced salt solution were packaged in sealable bags and exposed to 100–600 megapascals (MPa; 1 MPa = 10 atm = 147 psi) for 60 s in a commercial HPP unit. Positive (no HPP) and negative (No HPP but frozen/thawed) controls were examined in each experiment. None of the HPP untreated and frozen eggs (negative controls) were able to hatch or exclude trypan blue when placed in the hatching solution in experiment 1 or 2. HPP untreated and nonfrozen eggs (positive controls) hatched and excluded trypan blue; 75% were positive in experiment 1 and 80% were positive in experiment 2. No hatched eggs were observed when they were exposed to 300–600 MPa for 60 s. Treatment at 400 MPa and above caused rupturing of the oncosphere. Results from this study indicate that HPP is a possible method to inactivate tapeworm eggs and that the susceptibility of tapeworm eggs to HPP is similar to or greater than that of nematode eggs or tissue larvae. © 2010 Elsevier B.V. All rights reserved.
1. Introduction. High pressure processing (HPP) is an effective nonthermal means of eliminating nonspore-forming bacteria from a variety of food products (Flick, 2003). The shelf life of the products is extended and the sensory features (appearance, texture, taste, smell, etc.) of the food are not, or are only minimally, affected by HPP. Other advantages of HPP
∗ Corresponding author. Tel.: +1 540 231 6302. fax: +1 540 231 3426. E-mail address: [email protected] (D.S. Lindsay). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.10.055
over traditional thermal processing include reduced processing times; minimal heat damage problems; retention of freshness, flavor, texture, color and no vitamin C loss (Tewari et al., 1999). Little has been published on the effects of HPP on zoonotic metazoan parasites. Ohnishi et al. (1992, 1994) determined that pressures of greater than 200 megapascals (MPa; 1 MPa = 10 atm = 147 psi) kill 8-wk-old Trichinella spiralis larvae. Gamble et al. (1998) determined 55–60 MPa did not kill all T. spiralis larvae in pork tenderloin or diaphragm. Treatment at 200 MPa for 10 min at temperatures between 0 and 15 ◦ C kills Anisakis simplex larvae with
186
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Table 1 Effects of high hydrostatic pressure (HPP) on hatching of eggs of Hymenolepis diminuta exposed to no or 100–600 megapascals (MPa) pressure for 60 s. Pressure
% Hatched experiment 1
% Hatched experiment 2
Mean
0 MPa, unfrozen 0 MPa, frozen 100 MPa 200 MPa 300 MPa 400 MPa 500 MPa 600 MPa
75 0 18 16 0 0 0 0
80 0 19 15 0 0 0 0
77.5 0 18.5 15.0 0 0 0 0
lack of motility used as an indicator of larval death (MolinaGarcia and Sanz, 2002). Dong et al. (2003) found the times and pressures required to kill 100% of A. simplex larvae in king salmon and arrowtooth flounder were 30–60 s at 414 MPa, 90–180 s at 276 MPa and 180 s at 207 MPa. Rosypal et al. (2007) have demonstrated that unembryonated Ascaris suum eggs fail to develop after exposure to pressures of 241 MPa or more for 60 s, or when exposed to 276 MPa for 10–30 s. Hymenolepis diminuta is a rat tapeworm parasite with beetle intermediate hosts. It is commonly found in beetles in areas used for grain storage or storage of other dry foods. Humans, usually children are accidental hosts of H. diminuta and become infected through ingestion of cysticercoids in infected beetles. H. diminuta infection has sporadically been reported in humans from developed nations (Edelman et al., 1965; Levi et al., 1987; Hamrick et al., 1990) but is more frequent in developing nations (Jones, 1979; Weisse and Raszka, 1996; Wiwanitkit, 2004; Patamia et al., 2010). Eggs of the important zoonotic tapeworms Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption (Dorny et al., 2009; Eckert and Deplazes, 2004). Nothing is known about the susceptibility of tapeworm eggs to HPP treatment. Therefore, the present study was conducted using H. diminuta eggs collected from experimentally infected rats. 2. Materials and methods 2.1. Hymenolepis diminuta eggs H. diminuta eggs were collected from the feces of experimentally infected white rats, Rattus norvegicus and concentrated by flotation in Sheather’s sugar solution. Isolated eggs were stored in Hanks balanced salt solution (HBSS) at 4 ◦ C until used. Prior to use, 1 × 103 eggs in 1 ml HBSS were placed in sealable bags (Kapak Sealpak pouches, Minneapolis, Minnesota, USA) and the bags compressed to force out air. The bags were then sealed with a sealing machine (Shop sealer model FS-315, Fugi Impulse Vietnam Co. Ltd., Sai Gon-Linh Trung EPZ, Japan). The bags were placed in additional sealable bags and sealed. This additional bag was used to prevent any potential contamination of the HPP unit. Control eggs were prepared in bags but were not subjected to HPP. The positive control egg containing bags were kept at 4 ◦ C in refrigerator until used for hatching experiments. Bags containing H. diminuta eggs to be killed
and used as negative controls were subjected to several freeze–thaw cycles by freezing in a liquid nitrogen tank and thawing at room temperature. These negative control (dead) eggs were kept at 4 ◦ C in refrigerator until used for hatching experiments. 2.2. High pressure processing The sealed bags containing H. diminuta eggs were placed into a commercial HPP unit (Quintus Food Press QFP 35L-600 Model, Flow International Corporation [Avure Technologies], Kent, Washington, USA) with 7XS-6000intensifier pump and maximum operating pressure of 600 MPa. The HPP unit was installed and operated at Virginia Tech’s Department of Food Science and Technology. All samples were exposed for 60 s. Pressures of 100, 200, 300, 400, 500, and 600 MPa were examined and two experiments were conducted (Table 1). 2.3. Egg hatching assay The egg-hatching assay described by Allen et al. (1965) was used. Briefly, after HPP treatment, the processed H. diminuta egg solution was aspirated into 15 ml conical centrifuge tubes and 0.5 ml Tyrode’s solution containing 2 mm glass beads (N = 20) was added to each tube. The tubes were vortexed for 15–30 s to disrupt or remove the egg-shell. The tubes were centrifuged for 10 min and the supernatant and glass beads were removed. The sediment in each centrifuged tube was placed in a new 15 ml conical centrifuge tube and 14 ml of hatching solution (50 mg ␣-amylase, 0.3 mg trypsin in 40 ml Tyrode’s solution) was added and the sample vortexed 15–30 s. The solution was incubated at room temperature for 30 min. The tubes were centrifuged and the sediment was resuspended in 2 ml of a mixture of trypan blue in HBSS (1.8 ml HBSS + 0.2 ml 0.4% trypan blue solution) to determine viability of the hatched oncospheres. Oncospheres that stained blue were considered nonviable. Examinations were done using an Olympus BH60 microscope equipped with differential contrast optics (Olympus America Inc., Center Valley, Pennsylvania, USA). A total of 100 HPP treated or controls eggs were counted in 100 l aliquots and categorized as viable or nonviable for each treatment. 3. Results H. diminuta eggs not frozen and not treated with HPP and exposed to hatching medium were viable and the hex-
A.M.A. Merwad et al. / Veterinary Parasitology 176 (2011) 185–188
Fig. 1. Positive control Hymenolepis diminuta egg, not frozen and not treated with high hydrostatic pressure, hatching from egg placed in hatching medium. The hexacanth embryo (H) has exited through breaks in the inner (white arrowhead) and outer layers (arrow) of the eggshell. Cytoplasm (C) surrounds the hexacanth embryo and hooks (black arrowhead) are visible. Bar = 10 microns.
acanth embryo exited the eggs via a break in the eggshell (Fig. 1). Freezing in liquid nitrogen rendered 100% of the negative control eggs and various percentages of eggs treated at 100 or 200 MPa nonviable as judged by trypan blue exclusion (Fig. 2). No viable H. diminuta eggs were seen in samples treated with 300, 400, 500, or 600 MPa (Table 1). Treatment with these pressures induced breakage of the onchosphere wall and leakage of its contents in to the medium (Fig. 3). This was not observed in negative control (frozen) eggs.
Fig. 2. Nonviable negative control Hymenolepis diminuta treated with 200 MPa. The cytoplasm has taken up the trypan blue. Note the hooks arrows (arrows). Bar = 10 microns.
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Fig. 3. Hymenolepis diminuta egg treated with 500 MPa high hydrostatic pressure and demonstrating abnormal appearance. The covering (arrowhead) of the embryo has been disrupted and contents are leaking (arrow) into hatching medium. One pair of hooks (white arrowhead) has moved through an apparent disruption in the parasite covering membrane while the other two pairs of hooks remain inside the covering membrane (white arrows). Bar = 10 microns.
4. Discussion The effects of HPP treatment on the viability and structure of several protozoan parasites have been examined. Cryptosporidium parvum oocysts (Slifko et al., 2000; Collins et al., 2005), Encephalitozoon cuniculi spores (Jordan et al., 2005), Toxoplasma gondii oocysts (Lindsay et al., 2005, 2008) and tissue cysts (Lindsay et al. 2006), and Eimeria acervulina oocysts (Kniel et al., 2007) have been tested for the efficacy of HPP to inactivate the infectivity of these protozoans. Slifko et al. (2000) examined the effects of 550 MPa on C. parvum oocysts in apple and orange juice. They determined that a 60 s exposure at 550 MPa was 100% effective in decreasing infectivity of oocysts for cell cultures. The effects of HPP on the infectivity of C. parvum oocysts recovered from experimentally exposed HPP treated oysters for neonatal mice were examined by Collins et al. (2005). They found that a 180 s exposure to 550 MPa produced the maximum decrease in numbers of C. parvum positive mouse pups (93%) (Collins et al., 2005). Neither study (Slifko et al., 2000; Collins et al., 2005) examined the structural effects induced by HPP treatment on C. parvum oocysts. Jordan et al. (2005) examined the effects of HPP treatment of E. cuniculi spores on subsequent development in vitro. Spores treated with 200 to 275 MPa for 60 s had reduced infectivity for host cells. After treatment of 345 MPa or more for 60 s, spores were unable to infect host cells (Jordan et al., 2005). No structural alterations were observed in spores when they were examined using transmission electron microscopy after treatment with up to 480 MPa for 60 s min (Jordan et al., 2005). T. gondii oocysts in HBSS, dis-
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tilled water or spot-inoculated onto raspberries and treated with HPP for 60 s at ≥340 MPa were rendered noninfectious for mice (Lindsay et al., 2005, 2008). No structural alterations occurred in these oocysts using light microscopy. Tissue cysts of T. gondii in ground pork were rendered noninfectious for mice by treatment with ≥300 MPa for 30–90 s (Lindsay et al., 2006). They were not examined for structural alterations. Oocysts of E. acervulina collected from experimentally contaminated raspberries and basil treated with 550 MPa at 40 ◦ C for 2 min were rendered noninfectious for 3-wk-old chickens (Kniel et al., 2007). Adverse affects on the oocyst wall were not a cause for the loss of infectivity because the oocysts retained the ability to exclude methylene blue (Kniel et al., 2007) Rosypal et al. (2007) reported that no morphological alterations were noted with light microscopy when unembryonated A. suum eggs treated with 552 MPa for 60 s were compared to untreated controls. Ohnishi et al. (1994) examined the histochemical and morphological effects of HPP (10 min at 25 ◦ C) on T. spiralis in muscle tissues from experimentally infected mice after the tissue had been paraffin embedded and sections processed using Azan, periodic acid-Schiff (PAS), and routine hematoxylin and eosin (H&E) staining. At pressures of ≤100 MPa T. spiralis larvae stained normally. Alteration in larval structure associated with decreased Azan staining of stichocytes was observed at ≥150 MPa. They attributed this to acidiophilic structures becoming basophilic after HPP (Ohnishi et al., 1994). They also note decreased distortion of PAS positive staining of stichocytes treated with 300 MPa. They suggested that glycogen and/or glycoprotein’s demonstrated by PAS staining may decompose after HPP (Ohnishi et al., 1994). Acknowledgements The contribution of AMAM was supported by Partner and Ownership grant belonging to Ministry of Higher Education and Scientific Research, Egypt. This study was financially supported in part by an Animal Health and Disease grant to DSL and GJF. We thank Laura S. Douglas for her help with HPP treatment operations. References Allen, K.I., Bentzen, I., Voge, M., 1965. In vitro hatching of onchospheres of four hymenolepid cestodes. J. Parasitol. 51, 235–242. Collins, M.V., Flick, G.J., Smith, S.A., Fayer, R., Croonenberghs, R., O’Keefe, S., Lindsay, D.S., 2005. The effect of high-pressure processing on infectivity of Cryptosporidium parvum oocysts recovered from experimentally exposed Eastern oysters (Crassostrea virginica). J. Eukaryot. Microbiol. 52, 500–504.
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