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Effect of heat treatment on viability of Taenia hydatigena eggs
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Contents lists available at SciVerse ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
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Research Brief
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Effect of heat treatment on viability of Taenia hydatigena eggs
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Birpal S. Buttar a, Mark L. Nelson b,⇑, Jan R. Busboom a, Dale D. Hancock c, Douglas B. Walsh d, Douglas P. Jasmer e a
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Department of Animal Sciences, Washington State University, Pullman, WA 99164-6351, USA Department of Animal Sciences, P.O. Box 6351, 235 Clark Hall, Washington State University, Pullman, WA 99164-6351, USA c Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164-7010, USA d Department of Entomology, Washington State University, Pullman, WA 99164-6382, USA e Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA b
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" Heat treatment of Taenia hydatigena
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eggs for 5 min. " In vitro and in vivo analysis of viabilities. " In vitro reduction in viability of 99.47% at 60.00 °C. " In vivo reduction in viability of 100.00% at 57.38 °C.
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IN VITRO EXPERIMENT
IN VIVO EXPERIMENT
Slaughter at day 62 / 64
Ex-shelling, Activation Heat treated eggs
Active / Inactive Oncospheres
Infection with heat treated eggs
10%
Cysticerci recovered
30%
6% 2% -2% 20
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Temperature (°C)
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Cysticerci Recovered
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g r a p h i c a l a b s t r a c t
Percent Activation
h i g h l i g h t s
1 4 4 2 15
20% 10% 0% 0
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Temperature (°C)
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a r t i c l e 2 3 8 9 29 30 31 32 33
i n f o
Article history: Received 14 December 2011 Received in revised form 19 December 2012 Accepted 3 January 2013 Available online xxxx
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Keywords: Taenia hydatigena Cysticercosis Heat treatment
a b s t r a c t Effects of heat treatments on activation and infectivity of Taenia hydatigena eggs were assessed. Eggs containing oncospheres were used for in vitro and in vivo studies to determine the response to 5 min of heat treatment, ranging from room temperature (22 °C) to 60 °C. The study demonstrated 99.47% and 100% reduction in oncosphere activation or infectivity after 5 min of heat treatment at 60 °C and 57.38 °C under in vitro and in vivo conditions, respectively. Similar results between the two approaches indicted the appropriateness of the in vitro methods to identify oncosphericidal treatments of practical significance. Similar heat treatments may also be effective against Taenia saginata and help to reduce occurrence of beef cysticercosis. Ó 2013 Published by Elsevier Inc.
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1. Introduction Taenia saginata is a tapeworm of humans that causes cysticercosis in intermediate hosts (cattle) and taeniosis in definitive hosts (humans). Being a public health concern, strict post mortem regulations apply at the harvest level. Infected carcasses are often condemned resulting in large economic losses to beef producers.
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Abbreviations: PBS, phosphate buffered saline; ANCOVA, analysis of covariance. ⇑ Corresponding author. Fax: +1 509 335 4247. E-mail addresses: [email protected] (B.S. Buttar), [email protected] (M.L. Nelson), [email protected] (J.R. Busboom), [email protected] (D.D. Hancock), [email protected] (D.B. Walsh), [email protected] (D.P. Jasmer).
Cattle become infected orally; the most likely sources are thought to involve contaminated water and feed stuffs. Depending on the extent of exposure, the disease may be endemic or epidemic in cattle (Slonka et al., 1975, 1978; Fertig and Dorn, 1985; Weedon, 1987; Hancock et al., 1989; Yoder et al., 1994; Lees et al., 2002; Scandrett and Gajadhar, 2004). Based on 2006 United States Department of Agriculture Food Safety Inspection Service data obtained through Freedom of Information Act, the prevalence of beef cysticercosis was 0.067% (720 cases) in the Pacific Northwest compared to 0.004% (256 cases) in the entire USA. Further, prevalence of beef cysticercosis in the Pacific Northwest has been consistently higher than the rest of the USA at least since 1984. Heat treatment
0014-4894/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exppara.2013.01.004
Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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of wet feedstuffs is one possible method to eliminate viable Taenia spp. eggs. Measurements used by different investigators in in vitro and in vivo studies to assess viability of Taenia spp. eggs include staining, oncospheres activation analysis, culture, infection of intermediate hosts and lab animals (Williams and Colli, 1970; Wang et al., 1997; Minozzo et al., 2002; Chapalamadugu, 2006; Kyngdon et al., 2006; Chapalamadugu et al., 2008). Williams and Colli (1970) studied effect of heat on infectivity of Taenia hydatigena by inoculating heat treated ex-shelled and activated oncospheres into jirds (Meriones unguiculatus) but concluded that this in vivo system was unreliable. Chapalamadugu et al. (2008) concluded that accurate assessment of viability of Taenia taeniaeformis eggs using vital dyes was questionable after heat treatment and ex-shelling with 0.5% sodium hypochlorite. In vivo studies may yield the most reliable results provided that the intermediate and definitive hosts are available for the target species or an appropriate surrogate species is selected. For this study, activation or infectivity of oncospheres in eggs were evaluated in vitro or in vivo, respectively. Due to low availability of Taenia saginata, T. hydatigena was used as a surrogate species. T. hydatigena has a close phylogenetic relationship with T. saginata (Hoberg et al., 2001). A major difference between life cycles of T. hydatigena and T. saginata is the site where cysticerci localize in the intermediate host. T. hydatigena cysticerci show preference for the liver through which they migrate to reach the abdominal cavity (Soulsby, 1982). To a lesser extent, larvae may reach other tissues such as lungs (Blazek et al., 1985). In contrast, T. saginata cysticerci localize in skeletal and cardiac muscles. The objective of this study was to evaluate effect of heat treatments on activation and infectivity of T. hydatigena oncospheres in eggs.
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2. Materials and methods
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2.1. T. hydatigena eggs
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T. hydatigena eggs were obtained from Dr. M.W. Lightowlers (University of Melbourne, Australia). The parasite was then passaged at our facilities using sheep (intermediate host) and dogs (definitive host).
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2.2. Animals
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All research activities involving host animals (lambs and dogs) were approved by the Washington State University-Institutional Animal Care and Use Committee. Throughout the study the housing, environmental and nutritional quality for animal care met or exceeded FASS (2010) standards. All lambs in the study were fed starter diet (containing 36% steam rolled corn, 60% dehydrated alfalfa and 4% minerals, vitamins and protein supplements) for one week; grower diet (containing 52% steam rolled corn, 40% dehydrated alfalfa and 8% minerals, vitamins and protein supplements) for 3 weeks; and then finisher diet (containing 76% steam rolled corn, 20% alfalfa and 4% minerals, vitamins and protein supplements) until necropsy. Transitions between diets were carried out over a period of one week. All dietary formulations met or exceeded National Research Council requirements (NRC, 2007). Lambs were housed indoors in a temperature-controlled room (19–21 °C) with 13 h of light per day. Two dogs were used as the definitive host for the entire study. One dog was a 6 year old castrated red-tick coonhound, and the other was a 7 year old castrated blue-tick coonhound. The dogs were fed a commercial adult dog maintenance diet (2021 Teklad global 21% protein dog diet, Harlan laboratories, USA) and kept in temperature controlled (19–21 °C) indoor kennels with 13 h of light per day.
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2.3. Infections
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Three recently weaned lambs were used for each passage of T. hydatigena eggs. Lambs were orally infected with 100–2000 eggs, depending on the purpose of infection (parasite passage or for experiments). Eggs were administered orally to lambs in 2 ml phosphate buffered saline (PBS, pH = 7.2). The first batch of lambs was infected with eggs imported from Australia and slaughtered 56 days after infection. Thereafter, eggs were obtained from dogs that were infected orally with cysticerci obtained from infected lambs. Non-viable (calcified) and viable (non-calcified) cysticerci were recovered from omentum, liver surface, peritoneal cavity, diaphragm and lungs of lambs. Non-calcified cysticerci were collected in PBS. Infections in each of the two dogs were orally initiated with up to six of these cysticerci within an hour of collection. The dogs began shedding proglottids at 7 or 9 weeks after infection with cysticerci from the lambs, similar to results of Gregory (1976). The proglottids shed by dogs were found motile and were recovered from the floor or occasionally from feces. Up to six proglottids were recovered per dog each day and were placed into 1% Antibiotic–Antimycotic solution (Sigma Chem., USA) in PBS and stored at 4 °C. Active contractions of the proglottids expelled most of the eggs out into the solution within a few hours of collection. The eggs were stored at 4 °C and were used within one month of collection for in vitro or in vivo experiments. The dose of eggs necessary to provide an acceptable assessment of experimental treatments in lambs was determined in a preliminary experiment in which 100, 1000 or 2000 eggs were administered orally to lambs in 2 ml PBS. No morbidity or mortality was observed in any of the groups, with a mean cysticerci recovery rate of 10% of eggs administered initially. Thus, an acceptable infective dose of eggs was determined to be 2000 eggs per lamb for purposes of our experiments.
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2.4. Effect of heat treatment on in vitro oncosphere activation
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2.4.1. Design of activation experiments In a completely randomized design, temperature treatments were carried out in duplicate at 40, 45, 50, 55 and 60 °C for 5 min each. Heat treatments were selected based on a preliminary experiment in which 5 min of heat treatments of T. hydatigena eggs at 40, 50 and 60 °C resulted in activation rates of 5.88%, 2.38% and 0.00%. An untreated control was also maintained at room temperature of 22 °C. Egg concentrations were determined by counting eggs in 10 ll of the egg solution on a ceramic ring slide (Ring Microflocculation Slide, Clay Adams, USA) with a cover slip under a 400 magnification of a light microscope (BH-2, Olympus, USA). Four thousand eggs were diluted to a final volume of 1.2 ml with PBS in 2.0 ml flat bottom micro-centrifuge tubes for each treatment. Two heat treatments were carried out at a time using two heat blocks (VWR, Univar, USA), and temperature was monitored with a real-time digital scanning thermometer (12 Channel Scanning Benchtop Thermometer, Digi-Sense, Cole-Parmer Instrument Company, USA). A temperature probe was inserted into a micro centrifuge tube containing 1.2 ml PBS with no eggs. The 5 min heating time started when the temperature reached 40, 45, 50, 55 or 60 °C.
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2.4.2. Assessment of oncosphere activation Following heat treatment, ex-shelling was carried out to remove the outer embryophore of eggs, using a sodium hypochlorite technique with modifications (Lightowlers et al., 1984; Wang et al., 1997; Chapalamadugu et al., 2008). Sodium hypochlorite (0.3 ml of 6% NaOCl) was added to the heat-treated egg solution to a final concentration of 1.2%. The solution was shaken vigorously using
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Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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a vortex mixer (SP Deluxe Mixer – S8220, American Scientific Products, and USA) to facilitate disintegration of keratin blocks that envelop eggs. After 5 min, the solution was diluted to 2 ml with PBS to dilute the sodium hypochlorite to 0.9% and then washed thrice by centrifuging at 1000g for 10 min (Galaxy 7 Microcentrifuge, VWR international, USA); each time removing 1.6 ml supernatant and diluting to 2 ml with PBS. After the last wash, the supernatant was removed to retain a volume of 100 ll of ex-shelled oncosphere suspension. Sheep bile, collected at time of slaughter of lambs, was used for activation of oncospheres. Bile was collected from gall bladders using a syringe, pooled and stored at 20 °C in 1.5 ml microcentrifuge tubes. For activation, bile was thawed in a water bath at 37 °C and was then added (100 ll) to the solution of ex-shelled oncospheres (100 ll) to final bile concentration of 50%. This solution was incubated in a water bath (Water Bath 183, Precision Scientific Inc., USA) at 37 °C for 2 h, and then oncosphere activation was measured. To observe activation, 20 ll of the solution was transferred onto a ceramic ring slide and a cover slip placed on it. Eggs in each field were individually observed for movement of hooks and contractions (activation) under a light microscope (BH-2, Olympus, USA) at 400 magnification. Percentage of oncospheres demonstrating activity was then determined. Two hours of incubation in bile was selected based on results of a preliminary study in which maximum percent activity of oncospheres was observed at 2 h of incubation compared to other time periods of 0, 0.5, 1, 4, and 8 h of incubation. Gallie and Sewell (1970) reported that increased carbon dioxide in hatching medium improved activation rates of T. saginata oncospheres, but Ishiwata et al. (1993) found carbon dioxide reduced activation rates of T. taeniaeformis oncospheres. The effect of carbon dioxide may be a species-specific effect but was unknown for T. hydatigena. In preliminary experiments we noted no effect of carbon dioxide on activation rates of T. hydatigena oncospheres and thus, carbon dioxide was not used in our subsequent experiments. Heat treatment and exposure to sodium hypochlorite may lead to decreased oncosphere recoveries at higher temperatures, as suggested by Chapalamadugu et al. (2008) with T. taeniaeformis at temperatures above 65 °C. Therefore the percent recovery, based on an initial egg count of 4000, was determined and compared among treatments.
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2.5. Effect of in vivo heat treatments on infectivity of oncospheres
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There were three treatment groups in this experiment. Two heat treatments of eggs were carried out at 50 or 60 °C for 5 min each. Untreated control eggs were maintained at room temperature (22 °C). Following these treatments, three recently weaned Suffolk lambs (mean weight, 33.3 ± 1.07 kg) per treatment were infected orally, via syringe with 2000 eggs in 2 ml PBS. Lambs were weighed on day 0, 14, 21, 35, 49 and 56 post-infection to monitor any effect of treatment on weight gains. Five and four randomly selected lambs were slaughtered on days 62 and 64 post-infection, respectively. Captive bolt stunning was used for humane slaughter of lambs (Welty, 2007; FSIS, 2009). The numbers of cysticerci were counted for the omentum, liver surface, diaphragm, mesenteries, lungs and peritoneal cavity. Infectivity of oncospheres was calculated as percent of cysticerci recovered compared to eggs administered.
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2.6. Statistical analysis
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2.6.1. Analysis of in vitro activation experiments Two replicates per treatment were studied in a completely randomized design. A sigmoidal four-parameter model (Eq. (1)) (Rutledge, 2004; Chapalamadugu et al., 2008) was fitted using a nonlinear regression analysis of SigmaPlot 11.0 (SPSS Inc., Chicago,
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USA). Based on the same model, the four parameters were estimated using Proc NLIN of SAS 9.2 (SAS Institute, Cary, and North–Carolina, USA).
F ¼ y0 þ
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a 1 þ eð
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xx0 b
ð1Þ
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where f denotes the dependent variable – percent activity, x denotes independent variable – temperature treatment, and the four parameters are a (total change in percent activity), b (slope of the curve), x0 (inflection temperature) and y0 (minimum percent activity). To determine any interactions between heat treatment temperature and exposure to sodium hypochlorite, the oncosphere recoveries were compared at different temperatures using analysis of variance (Proc GLM of SAS 9.2).
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2.6.2. Analysis of in vivo infectivity experiments The percent of cysticerci recovered relative to eggs administered (viability) was regressed on temperature of heat treatment of eggs using linear regression (Proc REG of SAS 9.2). A split plot design was used to analyze the number of calcified, non-calcified and total cysticerci recovered from different sites of recovery, with temperature of egg treatment in the whole plot, individual lamb within temperature as the whole plot error term, site of recovery (organ) and its interactions were in the sub plot using Proc GLM of SAS 9.2. When a significant temperature site interaction was found, slice analysis was carried out, within temperature, for effect of sites of recovery on cysticerci count and within recovery site for effect of temperature on number of cysticerci recovered (Winer, 1971). The weights of lambs on day 0, 14, 21, 35, 49 and 56 post-infection were compared among treatments using ANCOVA with repeated measures and unequal time intervals using Proc MIXED of SAS 9.2.
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3. Results
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3.1. Effects of heat on in vitro activation of oncospheres
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Mean total oncospheres recovered did not significantly differ among heat treatments, indicating no detectable interaction between temperature and exposure to sodium hypochlorite, even though recovery was numerically lower after 60 °C treatment (Table 1). Maximum percent activation of recovered oncospheres was observed for treatment at 40 °C (6.14 ± 0.52%). Thereafter activation decreased with increased temperature to 60 °C, at which temperature no activation was observed (Table 1). Based on the data, the sigmoidal four-parameter model yielded the parameter values shown in Table 2 (R2 = 0.8718). This nonlinear model
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Table 1 In vitro effect of 5 min of thermal treatment on T. hydatigena eggs. Percent recovery of oncospheres ex-shelled with 1% sodium hypochlorite and percent activation of exshelled oncospheres treated with 50% bile treatments is shown. Temperature (°C)
Recovery ± SE (%)a
Activation ± SE (%)b
22 40 45 50 55 60
3.76 ± 0.46 3.07 ± 1.02 3.34 ± 0.74 2.37 ± 0.45 3.84 ± 0.66 1.51 ± 0.33
5.10 ± 0.96 6.14 ± 0.52 4.51 ± 1.63 1.92 ± 0.55 0.28 ± 0.28 0.00 ± 0.00
a The percent recovery was calculated based on egg count of subsample compared to the starting number of eggs used. b The percent activation was based on number of active oncospheres observed from the total number of ex-shelled oncospheres recovered. The effect of temperature was not significant regarding the percent of ex-shelled oncospheres recovered (p = 0.2139) and significant regarding percent activation (p < 0.0001).
Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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Table 2 Sigmoidal four-parameter model parameter estimates for in vitro percent activation of ex-shelled T. hydatigena eggs in response to heat treatment for 5 min at 22, 40, 45, 50, 55 and 60 °C temperatures. Parameter
Estimate
Standard error
95% Confidence Limits
a b x0 y0
5.6028 2.1571 48.5014 0.0028
1.0014 1.2750 1.5170 0.6931
3.2935 5.0973 45.0032 1.5954
predicted that percent activation would be reduced by 99.47% by heating at 60 °C for 5 min.
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3.2. Effects of heat treatments on in vivo infectivity of oncospheres
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The ANCOVA analysis of weights of lambs indicated no difference in slopes of the curves (weight gain per day) or intercepts (weight at day 0) among treatments. Average daily gain averaged 0.32 kg/day. The residuals of the count data for cysticerci recovered from different organs were normally distributed with skewness of 1.32 and kurtosis of 14.43. This slight deviation from a normal distribution may be explained by the excessive occurrence of zeros in the count data, after heat treatments at 50 and 60 °C (Table 3). Most of the calcified and non-calcified cysticerci were recovered from the omentum and the liver surface of lambs infected with eggs treated at 22 °C (control) or 50 °C, while no cysticerci were recovered from any site for lambs dosed with eggs heat treated at 60 °C (Table 3). The number of cysticerci that were observed on the liver surface (Fig. 1) was an excellent indicator of the treatment effects quantified in the Table 3. The mean percentage of cysticerci recovered were 11.55 ± 1.85%, 1.22 ± 0.54%, and 0.00 ± 0.00% of the total eggs administered for treatments at 22 (control), 50 and 60 °C, respectively. Linear regression analysis yielded the equation Y = 18.2490.318X (R2 = 0.877) where Y was the cysticerci recovered as percent of eggs administered and X was the temperature of heat treatment for 5 min (Fig. 2). The slope of the linear equation indicated mortality rate of 0.32% of total eggs administered per degree Celsius rise in temperature from 22 to 60 °C for 5 min. Additionally, the statistical model predicted 0% egg viability was attained at 57.38 °C. Split plot analysis of cysticerci (number of calcified, non-calcified and total number) recovered had significant interactions between the temperature of egg treatment and the site of recovery (Table 3). Subsequent slice analysis revealed that at 22 °C (control) the mean number of cysticerci recovered from omentum was
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Non-Calcified Cysticercus
b
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Calcified Cysticercus
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a = Total change in percent activity (%). b = Slope of the curve (%/°C). x0 = Inflection temperature (°C). y0 = Minimum percent activity (%).
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a
Calcified Cysticercus
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Fig. 1. Calcified cysticerci (non-viable) averaged 25.3, 9.3 and 0 and non-calcified cysterci (viable) averaged 10.7, 2.0 and 0 on the liver surfaces of lambs infected with T. hydatigena eggs heat treated at (a) 22 (control), (b) 50 and (c) 60 °C, respectively.
significantly different from all other sites for calcified, non-calcified and total cycticerci. However no such difference was observed at 50 °C. Also, the mean number of cysticerci recovered from omentum, liver, diaphragm and lungs decreased linearly with temperature of egg treatment. Mean recoveries from mesenteries and peritoneal cavity for all treatments were too low for a meaningful relationship between temperature and recovery. The ratio of calcified to non-calcified cysticerci was not affected by temperature of egg treatment or the tissue site of cysticerci recovery.
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4. Discussion
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In the present study, the effects of different heat treatments on the viabilities of T. hydatigena eggs were analyzed in vitro and in vivo. The most important observations were at 60 °C where no activation of oncospheres was observed in vitro and no cysticerci
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Table 3 Number of calcified (non-viable) and non-calcified (viable) cysticerci recovered from lambs infected with 2000 T. hydatigena eggs heat treated for 5 min at 22 (control), 50, 60 °C. Counts are the average number of cysticerci recovered from that organ. (SE = Standard error). Temperature of egg treatment for 5 min 22 °C (Control) Organ Number of cysticerci Omentumc Liverc Diaphragmc Lungsc Mesenteries Peri toneal cavity a,b c
50 °C
60 °C
Calcified SE = 12.86
Non-calcified SE = 8.08
Total SE = 17.04
Calcified SE = 2.91
Non-calcified SE = 0.96
Total SE = 3.56
Calcified SE = 0.00
Non-calcified SE = 0.00
Total SE = 0.00
124.0a 25.3b 5.7b 5.0b 0.7b 0.3b
54.3a 10.7b 2.7b 1.0b 0.3b 1.0b
178.3a 36.0b 8.3b 6.0b 1.0b 1.3b
10.0 9.3 0.0 0.0 0.0 0.0
2.7 2.0 0.0 0.3 0.0 0.0
12.7 11.3 0.0 0.3 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
Different superscripts within a column indicate difference among sites of recovery (p < 0.05) with slice analysis. Increasing temperature had a linear effect on total number of cysticerci recovered (p < 0.05) with slice analysis.
Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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Cysticerci recovered as percent of eggs administered (%)
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20% 18% 16% 14% 12% 10% 8% 6% 4% 2% 0% 0
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Temperature (°C)
Fig. 2. In vivo linear regression response of percent recovery of T. hydatigena cysticerci recovered as a percent of heat treated eggs administered at various temperatures (Y = 0. 318X + 18.249, R2 = 0.877).
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were recovered from lambs infected with eggs treated at this temperature. In vitro and in vivo statistical models established herein indicated a 99.47% and 100.00% reduction in viabilities after 5 min of heat treatment at 60.00 and 57.38 °C, respectively. The slope of the curve indicated mortality rates of oncospheres in response to increasing temperature was estimated at 2.25% and 0.32% of total oncospheres per degree Celsius increase for in vitro and in vivo models, respectively. Differences in results of in vitro and in vivo models may be in part due to use of different statistical approaches, e.g. a nonlinear sigmoidal four-parameter model for the in vitro experiment and a linear regression for the in vivo experiment. Nevertheless, it is also possible that the difference reflects greater sensitivity of the in vitro assay to detect viable eggs. For instance, there are many more potential factors affecting the eventual establishment of cysticerci following infection with oncospheres than is accounted for by the in vitro assays. More data points between 22 and 60 °C are recommended for future in vivo experiments to better fit a nonlinear regression model. When compared to the control group (22 °C), in vivo cysticerci recovery was reduced to 10% and 0% for 50 and 60 °C treatment groups, respectively. Similarly, the in vitro activation rates were 120, 88, 38, 5 and 0% of activation rates for control eggs (22 °C), for heat treated eggs at 40, 45, 50, 55 and 60 °C, respectively. Similar results were reported by Williams and Colli (1970) in that no in vitro activation of T. hydatigena oncospheres was observed after heat treatment at 55 °C for 5 min, 60 °C for 2 min and 65 °C for 1 min. Similarly, Allen (1947) predicted the thermal death point of T. saginata cysticerci to be 56 °C for 5 min. However, Pike (1988) and Hughes et al. (1985) cited by Bruce et al. (1990) reported a prolonged heat requirement (55 °C for 3 h) to reduce infectivity of T. saginata eggs to 1% in sludge. If the sludge had a higher coefficient of insulation, it may have protected the eggs for prolonged period. A similar situation may apply to potato coproducts. In the current study, mean percent active oncospheres recovered for egg treatment at 40 °C (5.98 ± 0.55%) was similar to egg treatment at 22 °C (4.98 ± 1.36%). This may indicate that heat treatment at 40 °C for 5 min has no detrimental effect on viability of T. hydatigena eggs. Moreover, all eggs in the in vitro study were incubated in bile at 37 °C for 2 h for activation, which further strengthens the possibility that 40 °C may not reduce viability of T. hydatigena eggs. In vitro ex-shelling and activation are important steps required to study viability of Taenia spp. The most widely adopted in vitro approaches have been ex-shelling with sodium hypochlorite
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(Lightowlers et al., 1984; Wang et al., 1997; Chapalamadugu et al., 2008) and activation with species-specific bile (Ishiwata et al., 1993; Wang et al., 1997; Kyngdon et al., 2006). For the current study, we used 1% sodium hypochlorite for ex-shelling and 50% sheep bile for activation in vitro. Viabilities of T. hydatigena eggs were not assessed using in vitro vital dye staining because accurate assessment of viability by staining with even low molecular weight dyes has been called into question (Chapalamadugu, 2006; Chapalamadugu et al., 2008). Instead activation rates were used to assess viability. Wang et al. (1997) reported activation rates of 4 and 1% and viabilities (based on trypan blue staining) of 80 and 87% for enzymatic and sodium hypochlorite ex-shelling techniques, respectively. All viable oncospheres may not activate at the same time, and this may explain the difference in percent activation rates and staining observed by Wang et al. (1997), but also dye exclusion appears to involve physical properties of the onchosperal membrane that are likely unrelated to onchosphere viability (Chapalamadugu et al., 2008). Different approaches have been suggested by many investigators to improve activation rates, but methods that yield consistent results remain unresolved. In the results presented here, maximum in vitro activation rates were about 6% after 2 h of incubation in bile. Although activation of all viable oncospheres may not have been observed at 2 h, similarity in results between the two approaches used here strengthens confidence that in vitro treatment effects are relevant to predictions on in vivo infectivity of oncospheres. Dead cysticerci in an intermediate host often calcify in response to the host’s immune system. The ratio of calcified to non-calcified cysticerci mainly depends on the host’s age, immune response and number of days since infection (Minozzo et al., 2002; Mehlhorn, 2006). The ratio of calcified to non-calcified cysts did not vary significantly among treatments in this investigation, which probably reflects similar age of hosts and timing of slaughter among the groups. This observation may mean that damage to oncospheres from heat treatment prevents early steps in the infection process, as compared to impacting long-term survival of cysticerci, once established in the host. Also, there was no effect of temperature of egg treatment or site of recovery (organ) on calcification status of cysticerci. Despite significant differences in cysticerci established among groups, no morbidity or mortality was observed in lambs used in the in vivo experiment. Mean rates of weight gain were also similar among treatment groups. These observations are consistent with the low pathogenicity of T. hydatigena infections in sheep (Soulsby, 1982). From an animal welfare perspective, this feature is another positive attribute of this model system. The results reported here present a promising foundation that can guide experiments to determine optimal heat treatment for inactivating T. saginata eggs. Accomplishing this goal has significant ramifications for public health and economical considerations to the potato processing and beef feedlot industries. In this regard, the relative unavailability of T. saginata eggs makes extensive research with this species problematic. Consequently, the availability of a well defined dog-sheep model for this human pathogen should help reach this goal. The model described can support further studies to better understand the effect of various time-temperature combinations of heat treatments without potato gelatinization. With optimal time and temperature treatments thus defined, targeted experiments with T. saginata eggs and the bovine host are conceivable to test predictions stemming from the T. hydatigena system. Use of a combination of methods for treating potato co-product may be required to fully render T. saginata eggs uninfective. For instance, lagooning of sludge at 7 °C for 28 days caused more than a 99% reduction in viability of T. saginata eggs (Bruce et al., 1990). We are investigating a similar approach involving ensilation of
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potato co-products, which occurs naturally during storage prior to feeding to cattle. Our data are encouraging on this point (manuscript in preparation). Identification of multiple treatments based on independent physical properties makes possible additive effects that are greater than either individual treatment alone, or more optimistically interactive effects that are greater than the combination of the two treatments alone. Significant progress is expected here towards defining approaches that will render potato co-products safe regarding infection of cattle with T. saginata. Importantly, current epidemiological systems are in place to assess the effectiveness of prevention programs resulting from this research (Yoder et al. 1994 and Hancock et al. 1989).
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Acknowledgments
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The study was supported by grants from Washington State Cattle Feeders Association, Conagra Foods–Lamb Weston and Risk Management Agency/Washington State University partnership project. We also thank Dr. Marshal William Lightowlers for supplying T. hydatigena eggs and consultation on experimental comport.
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References
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Allen, R.W., 1947. The thermal death point of cysticerci of Taenia saginata. J. Parasitol. 33, 331–338. Blazek, K., Schramlova, J., Hulinska, D., 1985. Pathology of the migration phase of Taenia hydatigena (Pallas, 1766) larvae. Folia Parasitol. (Praha) 32, 127–137. Bruce, A.M., Pike, E.B., Fisher, W.J., 1990. A review of treatment process options to meet the EC sludge directive. J. Inst. Water Environ. Manage. 4, 1–13. Chapalamadugu, K.C., 2006. Taenia taeniaeformis: differential staining on onchospheres with vital dyes under critical temperatures, MS Thesis. Washington State University, Pullman. Chapalamadugu, K.C., Busboom, J.R., Nelson, M.L., Hancock, D.D., Tang, J., Jasmer, D.P., 2008. Taenia taeniaeformis: effectiveness of staining oncospheres is related to both temperature of treatment and molecular weight of dyes utilized. Vet. Parasitol. 151, 203–211. FASS, 2010. Guide for the care and use of agricultural animals in research and teaching. Federation of Animal Science Societies, Champaign, Ill. Fertig, D.L., Dorn, C.R., 1985. Taenia saginata cysticercosis in an ohio cattle feeding operation. J. Am. Vet. Med. Assoc. 186, 1281–1285. FSIS, 2009. Humane Slaughter of Livestock. In: D.O.A., (Ed.), Food Safety and Inspection Service. Code of Federal Regulations, 9CFR313.15, Natl. Archiv. US, Washington, DC, USA. Gallie, G., Sewell, M., 1970. A technique for hatching Taenia saginata eggs. Vet. Rec. 86, 749. Gregory, G.G., 1976. Fecundity and proglottid release of Taenia ovis and T. hydatigena. Aust. Vet. J. 52, 277–279.
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Hancock, D.D., Wikse, S.E., Lichtenwalner, A.B., Wescott, R.B., Gay, C.C., 1989. Distribution of bovine cysticercosis in Washington. Am. J. Vet. Res. 50, 564–570. Hoberg, E.P., Alkire, N.L., de Queiroz, A., Jones, A., 2001. Out of Africa: origins of the Taenia tapeworms in humans. Proc. Biol. Sci. 268, 781–787. Hughes, D., Morris, D., Norrington, I., Waite, W., 1985. The Effects of Pasteurization and Stabilisation of Sludge on Taenia saginata Eggs. Elsevier Science Publishing Co., New York. Ishiwata, K., Oku, Y., Kamiya, M., 1993. The role of dissolved carbon-dioxide and whole bile in the in-vitro activation of Taenia taeniaeformis oncospheres. J. Helminthol. 67, 325–328. Kyngdon, C.T., Gauci, C.G., Rolfe, R.A., Velasquez Guzman, J.C., Farfan Salazar, M.J., Verastegui Pimentel, M.R., Gonzalez, A.E., Garcia, H.H., Gilmanl, R.H., Strugnell, R.A., Lightowlers, M.W., 2006. In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium. J. Parasitol. 92, 273–281. Lees, W., Nightingale, J., Brown, D., Scandrett, B., Gajadhar, A., 2002. Outbreak of Cysticercus bovis (Taenia saginata) in feedlot cattle in Alberta. Can. Vet. J. 43, 227–228. Lightowlers, M.W., Mitchell, G.F., Bowtell, D.D., Anders, R.F., Rickard, M.D., 1984. Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres. Int. J. Parasitol. 14, 321–333. Mehlhorn, H., 2006. Encyclopedic Reference of Parasitology. Springer, New York. Minozzo, J.C., Gusso, R.L.F., Castro, E.A.d., Lago, O., Soccol, V.T., 2002. Experimental bovine infection with Taenia saginata eggs: recovery rates and cysticerci location. Braz. Arch. Biol. Technol. 45, 451–455. NRC, 2007. Nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids. National Academies Press, Washington, DC. Pike, E.B., 1988. Infectivity of Taenia eggs after sludge treatment. WRc Report PRD 1199-M/2. M, Water Research Centre, p. 39. Rutledge, R.G., 2004. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications. Nucleic Acids Res. 32, e178. Scandrett, W.B., Gajadhar, A.A., 2004. Recovery of putative taeniid eggs from silt in water associated with an outbreak of bovine cysticercosis. Can. Vet. J. 45, 758– 760. Slonka, G.F., Matulich, W., Morphet, E., Miller, C.W., Bayer, E.V., 1978. An outbreak of bovine cysticercosis in California. Am. J. Trop. Med. Hyg. 27, 101–105. Slonka, G.F., Moulthrop, J.I., Dewhirst, L.W., Hotchkiss, P.M., Vallaza, B., Schultz, M.G., 1975. An epizootic of bovine cysticercosis. J. Am. Vet. Med. Assoc. 166, 678–681. Soulsby, E.J.L., 1982. Helminths, Arthropods, and Protozoa of Domesticated Animals. Lea & Febiger, Philadelphia. Wang, I.C., Ma, Y.X., Kuo, C.H., Fan, P.C., 1997. A comparative study on egg hatching methods and oncosphere viability determination for Taenia solium eggs. Int. J. Parasitol. 27, 1311–1314. Weedon, J.R., 1987. Cysticercosis. J. Am. Vet. Med. Assoc. 191, 1080–1081. Welty, J., 2007. Humane slaughter laws. Law Contemp. Probs. 70, 175. Williams, J.F., Colli, C.W., 1970. Primary Cystic Infection with Echinococcus granulosus and Taenia hydatigena in Meriones unguiculatus. J. Parasitol. 56, 509–513. Winer, B.J., 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York. Yoder, D.R., Ebel, E.D., Hancock, D.D., Combs, B.A., 1994. Epidemiologic findings from an outbreak of cysticercosis in feedlot cattle. J. Am. Vet. Med. Assoc. 205, 45–50.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
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Research Brief
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Effect of heat treatment on viability of Taenia hydatigena eggs
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Birpal S. Buttar a, Mark L. Nelson b,⇑, Jan R. Busboom a, Dale D. Hancock c, Douglas B. Walsh d, Douglas P. Jasmer e a
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Department of Animal Sciences, Washington State University, Pullman, WA 99164-6351, USA Department of Animal Sciences, P.O. Box 6351, 235 Clark Hall, Washington State University, Pullman, WA 99164-6351, USA c Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA 99164-7010, USA d Department of Entomology, Washington State University, Pullman, WA 99164-6382, USA e Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA b
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" Heat treatment of Taenia hydatigena
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eggs for 5 min. " In vitro and in vivo analysis of viabilities. " In vitro reduction in viability of 99.47% at 60.00 °C. " In vivo reduction in viability of 100.00% at 57.38 °C.
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IN VITRO EXPERIMENT
IN VIVO EXPERIMENT
Slaughter at day 62 / 64
Ex-shelling, Activation Heat treated eggs
Active / Inactive Oncospheres
Infection with heat treated eggs
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Cysticerci recovered
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Temperature (°C)
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Article history: Received 14 December 2011 Received in revised form 19 December 2012 Accepted 3 January 2013 Available online xxxx
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Keywords: Taenia hydatigena Cysticercosis Heat treatment
a b s t r a c t Effects of heat treatments on activation and infectivity of Taenia hydatigena eggs were assessed. Eggs containing oncospheres were used for in vitro and in vivo studies to determine the response to 5 min of heat treatment, ranging from room temperature (22 °C) to 60 °C. The study demonstrated 99.47% and 100% reduction in oncosphere activation or infectivity after 5 min of heat treatment at 60 °C and 57.38 °C under in vitro and in vivo conditions, respectively. Similar results between the two approaches indicted the appropriateness of the in vitro methods to identify oncosphericidal treatments of practical significance. Similar heat treatments may also be effective against Taenia saginata and help to reduce occurrence of beef cysticercosis. Ó 2013 Published by Elsevier Inc.
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1. Introduction Taenia saginata is a tapeworm of humans that causes cysticercosis in intermediate hosts (cattle) and taeniosis in definitive hosts (humans). Being a public health concern, strict post mortem regulations apply at the harvest level. Infected carcasses are often condemned resulting in large economic losses to beef producers.
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Abbreviations: PBS, phosphate buffered saline; ANCOVA, analysis of covariance. ⇑ Corresponding author. Fax: +1 509 335 4247. E-mail addresses: [email protected] (B.S. Buttar), [email protected] (M.L. Nelson), [email protected] (J.R. Busboom), [email protected] (D.D. Hancock), [email protected] (D.B. Walsh), [email protected] (D.P. Jasmer).
Cattle become infected orally; the most likely sources are thought to involve contaminated water and feed stuffs. Depending on the extent of exposure, the disease may be endemic or epidemic in cattle (Slonka et al., 1975, 1978; Fertig and Dorn, 1985; Weedon, 1987; Hancock et al., 1989; Yoder et al., 1994; Lees et al., 2002; Scandrett and Gajadhar, 2004). Based on 2006 United States Department of Agriculture Food Safety Inspection Service data obtained through Freedom of Information Act, the prevalence of beef cysticercosis was 0.067% (720 cases) in the Pacific Northwest compared to 0.004% (256 cases) in the entire USA. Further, prevalence of beef cysticercosis in the Pacific Northwest has been consistently higher than the rest of the USA at least since 1984. Heat treatment
0014-4894/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exppara.2013.01.004
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of wet feedstuffs is one possible method to eliminate viable Taenia spp. eggs. Measurements used by different investigators in in vitro and in vivo studies to assess viability of Taenia spp. eggs include staining, oncospheres activation analysis, culture, infection of intermediate hosts and lab animals (Williams and Colli, 1970; Wang et al., 1997; Minozzo et al., 2002; Chapalamadugu, 2006; Kyngdon et al., 2006; Chapalamadugu et al., 2008). Williams and Colli (1970) studied effect of heat on infectivity of Taenia hydatigena by inoculating heat treated ex-shelled and activated oncospheres into jirds (Meriones unguiculatus) but concluded that this in vivo system was unreliable. Chapalamadugu et al. (2008) concluded that accurate assessment of viability of Taenia taeniaeformis eggs using vital dyes was questionable after heat treatment and ex-shelling with 0.5% sodium hypochlorite. In vivo studies may yield the most reliable results provided that the intermediate and definitive hosts are available for the target species or an appropriate surrogate species is selected. For this study, activation or infectivity of oncospheres in eggs were evaluated in vitro or in vivo, respectively. Due to low availability of Taenia saginata, T. hydatigena was used as a surrogate species. T. hydatigena has a close phylogenetic relationship with T. saginata (Hoberg et al., 2001). A major difference between life cycles of T. hydatigena and T. saginata is the site where cysticerci localize in the intermediate host. T. hydatigena cysticerci show preference for the liver through which they migrate to reach the abdominal cavity (Soulsby, 1982). To a lesser extent, larvae may reach other tissues such as lungs (Blazek et al., 1985). In contrast, T. saginata cysticerci localize in skeletal and cardiac muscles. The objective of this study was to evaluate effect of heat treatments on activation and infectivity of T. hydatigena oncospheres in eggs.
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2. Materials and methods
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2.1. T. hydatigena eggs
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T. hydatigena eggs were obtained from Dr. M.W. Lightowlers (University of Melbourne, Australia). The parasite was then passaged at our facilities using sheep (intermediate host) and dogs (definitive host).
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2.2. Animals
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All research activities involving host animals (lambs and dogs) were approved by the Washington State University-Institutional Animal Care and Use Committee. Throughout the study the housing, environmental and nutritional quality for animal care met or exceeded FASS (2010) standards. All lambs in the study were fed starter diet (containing 36% steam rolled corn, 60% dehydrated alfalfa and 4% minerals, vitamins and protein supplements) for one week; grower diet (containing 52% steam rolled corn, 40% dehydrated alfalfa and 8% minerals, vitamins and protein supplements) for 3 weeks; and then finisher diet (containing 76% steam rolled corn, 20% alfalfa and 4% minerals, vitamins and protein supplements) until necropsy. Transitions between diets were carried out over a period of one week. All dietary formulations met or exceeded National Research Council requirements (NRC, 2007). Lambs were housed indoors in a temperature-controlled room (19–21 °C) with 13 h of light per day. Two dogs were used as the definitive host for the entire study. One dog was a 6 year old castrated red-tick coonhound, and the other was a 7 year old castrated blue-tick coonhound. The dogs were fed a commercial adult dog maintenance diet (2021 Teklad global 21% protein dog diet, Harlan laboratories, USA) and kept in temperature controlled (19–21 °C) indoor kennels with 13 h of light per day.
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2.3. Infections
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Three recently weaned lambs were used for each passage of T. hydatigena eggs. Lambs were orally infected with 100–2000 eggs, depending on the purpose of infection (parasite passage or for experiments). Eggs were administered orally to lambs in 2 ml phosphate buffered saline (PBS, pH = 7.2). The first batch of lambs was infected with eggs imported from Australia and slaughtered 56 days after infection. Thereafter, eggs were obtained from dogs that were infected orally with cysticerci obtained from infected lambs. Non-viable (calcified) and viable (non-calcified) cysticerci were recovered from omentum, liver surface, peritoneal cavity, diaphragm and lungs of lambs. Non-calcified cysticerci were collected in PBS. Infections in each of the two dogs were orally initiated with up to six of these cysticerci within an hour of collection. The dogs began shedding proglottids at 7 or 9 weeks after infection with cysticerci from the lambs, similar to results of Gregory (1976). The proglottids shed by dogs were found motile and were recovered from the floor or occasionally from feces. Up to six proglottids were recovered per dog each day and were placed into 1% Antibiotic–Antimycotic solution (Sigma Chem., USA) in PBS and stored at 4 °C. Active contractions of the proglottids expelled most of the eggs out into the solution within a few hours of collection. The eggs were stored at 4 °C and were used within one month of collection for in vitro or in vivo experiments. The dose of eggs necessary to provide an acceptable assessment of experimental treatments in lambs was determined in a preliminary experiment in which 100, 1000 or 2000 eggs were administered orally to lambs in 2 ml PBS. No morbidity or mortality was observed in any of the groups, with a mean cysticerci recovery rate of 10% of eggs administered initially. Thus, an acceptable infective dose of eggs was determined to be 2000 eggs per lamb for purposes of our experiments.
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2.4. Effect of heat treatment on in vitro oncosphere activation
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2.4.1. Design of activation experiments In a completely randomized design, temperature treatments were carried out in duplicate at 40, 45, 50, 55 and 60 °C for 5 min each. Heat treatments were selected based on a preliminary experiment in which 5 min of heat treatments of T. hydatigena eggs at 40, 50 and 60 °C resulted in activation rates of 5.88%, 2.38% and 0.00%. An untreated control was also maintained at room temperature of 22 °C. Egg concentrations were determined by counting eggs in 10 ll of the egg solution on a ceramic ring slide (Ring Microflocculation Slide, Clay Adams, USA) with a cover slip under a 400 magnification of a light microscope (BH-2, Olympus, USA). Four thousand eggs were diluted to a final volume of 1.2 ml with PBS in 2.0 ml flat bottom micro-centrifuge tubes for each treatment. Two heat treatments were carried out at a time using two heat blocks (VWR, Univar, USA), and temperature was monitored with a real-time digital scanning thermometer (12 Channel Scanning Benchtop Thermometer, Digi-Sense, Cole-Parmer Instrument Company, USA). A temperature probe was inserted into a micro centrifuge tube containing 1.2 ml PBS with no eggs. The 5 min heating time started when the temperature reached 40, 45, 50, 55 or 60 °C.
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2.4.2. Assessment of oncosphere activation Following heat treatment, ex-shelling was carried out to remove the outer embryophore of eggs, using a sodium hypochlorite technique with modifications (Lightowlers et al., 1984; Wang et al., 1997; Chapalamadugu et al., 2008). Sodium hypochlorite (0.3 ml of 6% NaOCl) was added to the heat-treated egg solution to a final concentration of 1.2%. The solution was shaken vigorously using
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a vortex mixer (SP Deluxe Mixer – S8220, American Scientific Products, and USA) to facilitate disintegration of keratin blocks that envelop eggs. After 5 min, the solution was diluted to 2 ml with PBS to dilute the sodium hypochlorite to 0.9% and then washed thrice by centrifuging at 1000g for 10 min (Galaxy 7 Microcentrifuge, VWR international, USA); each time removing 1.6 ml supernatant and diluting to 2 ml with PBS. After the last wash, the supernatant was removed to retain a volume of 100 ll of ex-shelled oncosphere suspension. Sheep bile, collected at time of slaughter of lambs, was used for activation of oncospheres. Bile was collected from gall bladders using a syringe, pooled and stored at 20 °C in 1.5 ml microcentrifuge tubes. For activation, bile was thawed in a water bath at 37 °C and was then added (100 ll) to the solution of ex-shelled oncospheres (100 ll) to final bile concentration of 50%. This solution was incubated in a water bath (Water Bath 183, Precision Scientific Inc., USA) at 37 °C for 2 h, and then oncosphere activation was measured. To observe activation, 20 ll of the solution was transferred onto a ceramic ring slide and a cover slip placed on it. Eggs in each field were individually observed for movement of hooks and contractions (activation) under a light microscope (BH-2, Olympus, USA) at 400 magnification. Percentage of oncospheres demonstrating activity was then determined. Two hours of incubation in bile was selected based on results of a preliminary study in which maximum percent activity of oncospheres was observed at 2 h of incubation compared to other time periods of 0, 0.5, 1, 4, and 8 h of incubation. Gallie and Sewell (1970) reported that increased carbon dioxide in hatching medium improved activation rates of T. saginata oncospheres, but Ishiwata et al. (1993) found carbon dioxide reduced activation rates of T. taeniaeformis oncospheres. The effect of carbon dioxide may be a species-specific effect but was unknown for T. hydatigena. In preliminary experiments we noted no effect of carbon dioxide on activation rates of T. hydatigena oncospheres and thus, carbon dioxide was not used in our subsequent experiments. Heat treatment and exposure to sodium hypochlorite may lead to decreased oncosphere recoveries at higher temperatures, as suggested by Chapalamadugu et al. (2008) with T. taeniaeformis at temperatures above 65 °C. Therefore the percent recovery, based on an initial egg count of 4000, was determined and compared among treatments.
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2.5. Effect of in vivo heat treatments on infectivity of oncospheres
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There were three treatment groups in this experiment. Two heat treatments of eggs were carried out at 50 or 60 °C for 5 min each. Untreated control eggs were maintained at room temperature (22 °C). Following these treatments, three recently weaned Suffolk lambs (mean weight, 33.3 ± 1.07 kg) per treatment were infected orally, via syringe with 2000 eggs in 2 ml PBS. Lambs were weighed on day 0, 14, 21, 35, 49 and 56 post-infection to monitor any effect of treatment on weight gains. Five and four randomly selected lambs were slaughtered on days 62 and 64 post-infection, respectively. Captive bolt stunning was used for humane slaughter of lambs (Welty, 2007; FSIS, 2009). The numbers of cysticerci were counted for the omentum, liver surface, diaphragm, mesenteries, lungs and peritoneal cavity. Infectivity of oncospheres was calculated as percent of cysticerci recovered compared to eggs administered.
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2.6.1. Analysis of in vitro activation experiments Two replicates per treatment were studied in a completely randomized design. A sigmoidal four-parameter model (Eq. (1)) (Rutledge, 2004; Chapalamadugu et al., 2008) was fitted using a nonlinear regression analysis of SigmaPlot 11.0 (SPSS Inc., Chicago,
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USA). Based on the same model, the four parameters were estimated using Proc NLIN of SAS 9.2 (SAS Institute, Cary, and North–Carolina, USA).
F ¼ y0 þ
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a 1 þ eð
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xx0 b
ð1Þ
Þ
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where f denotes the dependent variable – percent activity, x denotes independent variable – temperature treatment, and the four parameters are a (total change in percent activity), b (slope of the curve), x0 (inflection temperature) and y0 (minimum percent activity). To determine any interactions between heat treatment temperature and exposure to sodium hypochlorite, the oncosphere recoveries were compared at different temperatures using analysis of variance (Proc GLM of SAS 9.2).
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2.6.2. Analysis of in vivo infectivity experiments The percent of cysticerci recovered relative to eggs administered (viability) was regressed on temperature of heat treatment of eggs using linear regression (Proc REG of SAS 9.2). A split plot design was used to analyze the number of calcified, non-calcified and total cysticerci recovered from different sites of recovery, with temperature of egg treatment in the whole plot, individual lamb within temperature as the whole plot error term, site of recovery (organ) and its interactions were in the sub plot using Proc GLM of SAS 9.2. When a significant temperature site interaction was found, slice analysis was carried out, within temperature, for effect of sites of recovery on cysticerci count and within recovery site for effect of temperature on number of cysticerci recovered (Winer, 1971). The weights of lambs on day 0, 14, 21, 35, 49 and 56 post-infection were compared among treatments using ANCOVA with repeated measures and unequal time intervals using Proc MIXED of SAS 9.2.
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3. Results
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3.1. Effects of heat on in vitro activation of oncospheres
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Mean total oncospheres recovered did not significantly differ among heat treatments, indicating no detectable interaction between temperature and exposure to sodium hypochlorite, even though recovery was numerically lower after 60 °C treatment (Table 1). Maximum percent activation of recovered oncospheres was observed for treatment at 40 °C (6.14 ± 0.52%). Thereafter activation decreased with increased temperature to 60 °C, at which temperature no activation was observed (Table 1). Based on the data, the sigmoidal four-parameter model yielded the parameter values shown in Table 2 (R2 = 0.8718). This nonlinear model
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Table 1 In vitro effect of 5 min of thermal treatment on T. hydatigena eggs. Percent recovery of oncospheres ex-shelled with 1% sodium hypochlorite and percent activation of exshelled oncospheres treated with 50% bile treatments is shown. Temperature (°C)
Recovery ± SE (%)a
Activation ± SE (%)b
22 40 45 50 55 60
3.76 ± 0.46 3.07 ± 1.02 3.34 ± 0.74 2.37 ± 0.45 3.84 ± 0.66 1.51 ± 0.33
5.10 ± 0.96 6.14 ± 0.52 4.51 ± 1.63 1.92 ± 0.55 0.28 ± 0.28 0.00 ± 0.00
a The percent recovery was calculated based on egg count of subsample compared to the starting number of eggs used. b The percent activation was based on number of active oncospheres observed from the total number of ex-shelled oncospheres recovered. The effect of temperature was not significant regarding the percent of ex-shelled oncospheres recovered (p = 0.2139) and significant regarding percent activation (p < 0.0001).
Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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Table 2 Sigmoidal four-parameter model parameter estimates for in vitro percent activation of ex-shelled T. hydatigena eggs in response to heat treatment for 5 min at 22, 40, 45, 50, 55 and 60 °C temperatures. Parameter
Estimate
Standard error
95% Confidence Limits
a b x0 y0
5.6028 2.1571 48.5014 0.0028
1.0014 1.2750 1.5170 0.6931
3.2935 5.0973 45.0032 1.5954
predicted that percent activation would be reduced by 99.47% by heating at 60 °C for 5 min.
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3.2. Effects of heat treatments on in vivo infectivity of oncospheres
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The ANCOVA analysis of weights of lambs indicated no difference in slopes of the curves (weight gain per day) or intercepts (weight at day 0) among treatments. Average daily gain averaged 0.32 kg/day. The residuals of the count data for cysticerci recovered from different organs were normally distributed with skewness of 1.32 and kurtosis of 14.43. This slight deviation from a normal distribution may be explained by the excessive occurrence of zeros in the count data, after heat treatments at 50 and 60 °C (Table 3). Most of the calcified and non-calcified cysticerci were recovered from the omentum and the liver surface of lambs infected with eggs treated at 22 °C (control) or 50 °C, while no cysticerci were recovered from any site for lambs dosed with eggs heat treated at 60 °C (Table 3). The number of cysticerci that were observed on the liver surface (Fig. 1) was an excellent indicator of the treatment effects quantified in the Table 3. The mean percentage of cysticerci recovered were 11.55 ± 1.85%, 1.22 ± 0.54%, and 0.00 ± 0.00% of the total eggs administered for treatments at 22 (control), 50 and 60 °C, respectively. Linear regression analysis yielded the equation Y = 18.2490.318X (R2 = 0.877) where Y was the cysticerci recovered as percent of eggs administered and X was the temperature of heat treatment for 5 min (Fig. 2). The slope of the linear equation indicated mortality rate of 0.32% of total eggs administered per degree Celsius rise in temperature from 22 to 60 °C for 5 min. Additionally, the statistical model predicted 0% egg viability was attained at 57.38 °C. Split plot analysis of cysticerci (number of calcified, non-calcified and total number) recovered had significant interactions between the temperature of egg treatment and the site of recovery (Table 3). Subsequent slice analysis revealed that at 22 °C (control) the mean number of cysticerci recovered from omentum was
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Non-Calcified Cysticercus
b
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Calcified Cysticercus
7.9121 0.7831 51.9996 1.6011
a = Total change in percent activity (%). b = Slope of the curve (%/°C). x0 = Inflection temperature (°C). y0 = Minimum percent activity (%).
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a
Calcified Cysticercus
c
Fig. 1. Calcified cysticerci (non-viable) averaged 25.3, 9.3 and 0 and non-calcified cysterci (viable) averaged 10.7, 2.0 and 0 on the liver surfaces of lambs infected with T. hydatigena eggs heat treated at (a) 22 (control), (b) 50 and (c) 60 °C, respectively.
significantly different from all other sites for calcified, non-calcified and total cycticerci. However no such difference was observed at 50 °C. Also, the mean number of cysticerci recovered from omentum, liver, diaphragm and lungs decreased linearly with temperature of egg treatment. Mean recoveries from mesenteries and peritoneal cavity for all treatments were too low for a meaningful relationship between temperature and recovery. The ratio of calcified to non-calcified cysticerci was not affected by temperature of egg treatment or the tissue site of cysticerci recovery.
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4. Discussion
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In the present study, the effects of different heat treatments on the viabilities of T. hydatigena eggs were analyzed in vitro and in vivo. The most important observations were at 60 °C where no activation of oncospheres was observed in vitro and no cysticerci
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Table 3 Number of calcified (non-viable) and non-calcified (viable) cysticerci recovered from lambs infected with 2000 T. hydatigena eggs heat treated for 5 min at 22 (control), 50, 60 °C. Counts are the average number of cysticerci recovered from that organ. (SE = Standard error). Temperature of egg treatment for 5 min 22 °C (Control) Organ Number of cysticerci Omentumc Liverc Diaphragmc Lungsc Mesenteries Peri toneal cavity a,b c
50 °C
60 °C
Calcified SE = 12.86
Non-calcified SE = 8.08
Total SE = 17.04
Calcified SE = 2.91
Non-calcified SE = 0.96
Total SE = 3.56
Calcified SE = 0.00
Non-calcified SE = 0.00
Total SE = 0.00
124.0a 25.3b 5.7b 5.0b 0.7b 0.3b
54.3a 10.7b 2.7b 1.0b 0.3b 1.0b
178.3a 36.0b 8.3b 6.0b 1.0b 1.3b
10.0 9.3 0.0 0.0 0.0 0.0
2.7 2.0 0.0 0.3 0.0 0.0
12.7 11.3 0.0 0.3 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0
Different superscripts within a column indicate difference among sites of recovery (p < 0.05) with slice analysis. Increasing temperature had a linear effect on total number of cysticerci recovered (p < 0.05) with slice analysis.
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Temperature (°C)
Fig. 2. In vivo linear regression response of percent recovery of T. hydatigena cysticerci recovered as a percent of heat treated eggs administered at various temperatures (Y = 0. 318X + 18.249, R2 = 0.877).
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were recovered from lambs infected with eggs treated at this temperature. In vitro and in vivo statistical models established herein indicated a 99.47% and 100.00% reduction in viabilities after 5 min of heat treatment at 60.00 and 57.38 °C, respectively. The slope of the curve indicated mortality rates of oncospheres in response to increasing temperature was estimated at 2.25% and 0.32% of total oncospheres per degree Celsius increase for in vitro and in vivo models, respectively. Differences in results of in vitro and in vivo models may be in part due to use of different statistical approaches, e.g. a nonlinear sigmoidal four-parameter model for the in vitro experiment and a linear regression for the in vivo experiment. Nevertheless, it is also possible that the difference reflects greater sensitivity of the in vitro assay to detect viable eggs. For instance, there are many more potential factors affecting the eventual establishment of cysticerci following infection with oncospheres than is accounted for by the in vitro assays. More data points between 22 and 60 °C are recommended for future in vivo experiments to better fit a nonlinear regression model. When compared to the control group (22 °C), in vivo cysticerci recovery was reduced to 10% and 0% for 50 and 60 °C treatment groups, respectively. Similarly, the in vitro activation rates were 120, 88, 38, 5 and 0% of activation rates for control eggs (22 °C), for heat treated eggs at 40, 45, 50, 55 and 60 °C, respectively. Similar results were reported by Williams and Colli (1970) in that no in vitro activation of T. hydatigena oncospheres was observed after heat treatment at 55 °C for 5 min, 60 °C for 2 min and 65 °C for 1 min. Similarly, Allen (1947) predicted the thermal death point of T. saginata cysticerci to be 56 °C for 5 min. However, Pike (1988) and Hughes et al. (1985) cited by Bruce et al. (1990) reported a prolonged heat requirement (55 °C for 3 h) to reduce infectivity of T. saginata eggs to 1% in sludge. If the sludge had a higher coefficient of insulation, it may have protected the eggs for prolonged period. A similar situation may apply to potato coproducts. In the current study, mean percent active oncospheres recovered for egg treatment at 40 °C (5.98 ± 0.55%) was similar to egg treatment at 22 °C (4.98 ± 1.36%). This may indicate that heat treatment at 40 °C for 5 min has no detrimental effect on viability of T. hydatigena eggs. Moreover, all eggs in the in vitro study were incubated in bile at 37 °C for 2 h for activation, which further strengthens the possibility that 40 °C may not reduce viability of T. hydatigena eggs. In vitro ex-shelling and activation are important steps required to study viability of Taenia spp. The most widely adopted in vitro approaches have been ex-shelling with sodium hypochlorite
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(Lightowlers et al., 1984; Wang et al., 1997; Chapalamadugu et al., 2008) and activation with species-specific bile (Ishiwata et al., 1993; Wang et al., 1997; Kyngdon et al., 2006). For the current study, we used 1% sodium hypochlorite for ex-shelling and 50% sheep bile for activation in vitro. Viabilities of T. hydatigena eggs were not assessed using in vitro vital dye staining because accurate assessment of viability by staining with even low molecular weight dyes has been called into question (Chapalamadugu, 2006; Chapalamadugu et al., 2008). Instead activation rates were used to assess viability. Wang et al. (1997) reported activation rates of 4 and 1% and viabilities (based on trypan blue staining) of 80 and 87% for enzymatic and sodium hypochlorite ex-shelling techniques, respectively. All viable oncospheres may not activate at the same time, and this may explain the difference in percent activation rates and staining observed by Wang et al. (1997), but also dye exclusion appears to involve physical properties of the onchosperal membrane that are likely unrelated to onchosphere viability (Chapalamadugu et al., 2008). Different approaches have been suggested by many investigators to improve activation rates, but methods that yield consistent results remain unresolved. In the results presented here, maximum in vitro activation rates were about 6% after 2 h of incubation in bile. Although activation of all viable oncospheres may not have been observed at 2 h, similarity in results between the two approaches used here strengthens confidence that in vitro treatment effects are relevant to predictions on in vivo infectivity of oncospheres. Dead cysticerci in an intermediate host often calcify in response to the host’s immune system. The ratio of calcified to non-calcified cysticerci mainly depends on the host’s age, immune response and number of days since infection (Minozzo et al., 2002; Mehlhorn, 2006). The ratio of calcified to non-calcified cysts did not vary significantly among treatments in this investigation, which probably reflects similar age of hosts and timing of slaughter among the groups. This observation may mean that damage to oncospheres from heat treatment prevents early steps in the infection process, as compared to impacting long-term survival of cysticerci, once established in the host. Also, there was no effect of temperature of egg treatment or site of recovery (organ) on calcification status of cysticerci. Despite significant differences in cysticerci established among groups, no morbidity or mortality was observed in lambs used in the in vivo experiment. Mean rates of weight gain were also similar among treatment groups. These observations are consistent with the low pathogenicity of T. hydatigena infections in sheep (Soulsby, 1982). From an animal welfare perspective, this feature is another positive attribute of this model system. The results reported here present a promising foundation that can guide experiments to determine optimal heat treatment for inactivating T. saginata eggs. Accomplishing this goal has significant ramifications for public health and economical considerations to the potato processing and beef feedlot industries. In this regard, the relative unavailability of T. saginata eggs makes extensive research with this species problematic. Consequently, the availability of a well defined dog-sheep model for this human pathogen should help reach this goal. The model described can support further studies to better understand the effect of various time-temperature combinations of heat treatments without potato gelatinization. With optimal time and temperature treatments thus defined, targeted experiments with T. saginata eggs and the bovine host are conceivable to test predictions stemming from the T. hydatigena system. Use of a combination of methods for treating potato co-product may be required to fully render T. saginata eggs uninfective. For instance, lagooning of sludge at 7 °C for 28 days caused more than a 99% reduction in viability of T. saginata eggs (Bruce et al., 1990). We are investigating a similar approach involving ensilation of
Please cite this article in press as: Buttar, B.S., et al. Effect of heat treatment on viability of Taenia hydatigena eggs. Exp. Parasitol. (2013), http://dx.doi.org/ 10.1016/j.exppara.2013.01.004
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potato co-products, which occurs naturally during storage prior to feeding to cattle. Our data are encouraging on this point (manuscript in preparation). Identification of multiple treatments based on independent physical properties makes possible additive effects that are greater than either individual treatment alone, or more optimistically interactive effects that are greater than the combination of the two treatments alone. Significant progress is expected here towards defining approaches that will render potato co-products safe regarding infection of cattle with T. saginata. Importantly, current epidemiological systems are in place to assess the effectiveness of prevention programs resulting from this research (Yoder et al. 1994 and Hancock et al. 1989).
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Acknowledgments
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The study was supported by grants from Washington State Cattle Feeders Association, Conagra Foods–Lamb Weston and Risk Management Agency/Washington State University partnership project. We also thank Dr. Marshal William Lightowlers for supplying T. hydatigena eggs and consultation on experimental comport.
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References
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Allen, R.W., 1947. The thermal death point of cysticerci of Taenia saginata. J. Parasitol. 33, 331–338. Blazek, K., Schramlova, J., Hulinska, D., 1985. Pathology of the migration phase of Taenia hydatigena (Pallas, 1766) larvae. Folia Parasitol. (Praha) 32, 127–137. Bruce, A.M., Pike, E.B., Fisher, W.J., 1990. A review of treatment process options to meet the EC sludge directive. J. Inst. Water Environ. Manage. 4, 1–13. Chapalamadugu, K.C., 2006. Taenia taeniaeformis: differential staining on onchospheres with vital dyes under critical temperatures, MS Thesis. Washington State University, Pullman. Chapalamadugu, K.C., Busboom, J.R., Nelson, M.L., Hancock, D.D., Tang, J., Jasmer, D.P., 2008. Taenia taeniaeformis: effectiveness of staining oncospheres is related to both temperature of treatment and molecular weight of dyes utilized. Vet. Parasitol. 151, 203–211. FASS, 2010. Guide for the care and use of agricultural animals in research and teaching. Federation of Animal Science Societies, Champaign, Ill. Fertig, D.L., Dorn, C.R., 1985. Taenia saginata cysticercosis in an ohio cattle feeding operation. J. Am. Vet. Med. Assoc. 186, 1281–1285. FSIS, 2009. Humane Slaughter of Livestock. In: D.O.A., (Ed.), Food Safety and Inspection Service. Code of Federal Regulations, 9CFR313.15, Natl. Archiv. US, Washington, DC, USA. Gallie, G., Sewell, M., 1970. A technique for hatching Taenia saginata eggs. Vet. Rec. 86, 749. Gregory, G.G., 1976. Fecundity and proglottid release of Taenia ovis and T. hydatigena. Aust. Vet. J. 52, 277–279.
455 456 457 458 459 460 461 462 463 464 465
469 470 471
Q3
Hancock, D.D., Wikse, S.E., Lichtenwalner, A.B., Wescott, R.B., Gay, C.C., 1989. Distribution of bovine cysticercosis in Washington. Am. J. Vet. Res. 50, 564–570. Hoberg, E.P., Alkire, N.L., de Queiroz, A., Jones, A., 2001. Out of Africa: origins of the Taenia tapeworms in humans. Proc. Biol. Sci. 268, 781–787. Hughes, D., Morris, D., Norrington, I., Waite, W., 1985. The Effects of Pasteurization and Stabilisation of Sludge on Taenia saginata Eggs. Elsevier Science Publishing Co., New York. Ishiwata, K., Oku, Y., Kamiya, M., 1993. The role of dissolved carbon-dioxide and whole bile in the in-vitro activation of Taenia taeniaeformis oncospheres. J. Helminthol. 67, 325–328. Kyngdon, C.T., Gauci, C.G., Rolfe, R.A., Velasquez Guzman, J.C., Farfan Salazar, M.J., Verastegui Pimentel, M.R., Gonzalez, A.E., Garcia, H.H., Gilmanl, R.H., Strugnell, R.A., Lightowlers, M.W., 2006. In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium. J. Parasitol. 92, 273–281. Lees, W., Nightingale, J., Brown, D., Scandrett, B., Gajadhar, A., 2002. Outbreak of Cysticercus bovis (Taenia saginata) in feedlot cattle in Alberta. Can. Vet. J. 43, 227–228. Lightowlers, M.W., Mitchell, G.F., Bowtell, D.D., Anders, R.F., Rickard, M.D., 1984. Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres. Int. J. Parasitol. 14, 321–333. Mehlhorn, H., 2006. Encyclopedic Reference of Parasitology. Springer, New York. Minozzo, J.C., Gusso, R.L.F., Castro, E.A.d., Lago, O., Soccol, V.T., 2002. Experimental bovine infection with Taenia saginata eggs: recovery rates and cysticerci location. Braz. Arch. Biol. Technol. 45, 451–455. NRC, 2007. Nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids. National Academies Press, Washington, DC. Pike, E.B., 1988. Infectivity of Taenia eggs after sludge treatment. WRc Report PRD 1199-M/2. M, Water Research Centre, p. 39. Rutledge, R.G., 2004. Sigmoidal curve-fitting redefines quantitative real-time PCR with the prospective of developing automated high-throughput applications. Nucleic Acids Res. 32, e178. Scandrett, W.B., Gajadhar, A.A., 2004. Recovery of putative taeniid eggs from silt in water associated with an outbreak of bovine cysticercosis. Can. Vet. J. 45, 758– 760. Slonka, G.F., Matulich, W., Morphet, E., Miller, C.W., Bayer, E.V., 1978. An outbreak of bovine cysticercosis in California. Am. J. Trop. Med. Hyg. 27, 101–105. Slonka, G.F., Moulthrop, J.I., Dewhirst, L.W., Hotchkiss, P.M., Vallaza, B., Schultz, M.G., 1975. An epizootic of bovine cysticercosis. J. Am. Vet. Med. Assoc. 166, 678–681. Soulsby, E.J.L., 1982. Helminths, Arthropods, and Protozoa of Domesticated Animals. Lea & Febiger, Philadelphia. Wang, I.C., Ma, Y.X., Kuo, C.H., Fan, P.C., 1997. A comparative study on egg hatching methods and oncosphere viability determination for Taenia solium eggs. Int. J. Parasitol. 27, 1311–1314. Weedon, J.R., 1987. Cysticercosis. J. Am. Vet. Med. Assoc. 191, 1080–1081. Welty, J., 2007. Humane slaughter laws. Law Contemp. Probs. 70, 175. Williams, J.F., Colli, C.W., 1970. Primary Cystic Infection with Echinococcus granulosus and Taenia hydatigena in Meriones unguiculatus. J. Parasitol. 56, 509–513. Winer, B.J., 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York. Yoder, D.R., Ebel, E.D., Hancock, D.D., Combs, B.A., 1994. Epidemiologic findings from an outbreak of cysticercosis in feedlot cattle. J. Am. Vet. Med. Assoc. 205, 45–50.
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