Survival of experimentally induced Toxoplasma gondii tissue cysts in vacuum packed goat meat and dry fermented goat meat sausages

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<ul><li><p>lable at ScienceDirect</p><p>Food Microbiology 39 (2014) 47e52Contents lists avaiFood Microbiology</p><p>journal homepage: www.elsevier .com/locate/ fmSurvival of experimentally induced Toxoplasma gondii tissue cysts invacuum packed goat meat and dry fermented goat meat sausages</p><p>Helena Neumayerov a,*, Jana Jurnkov a, Alena Salkov b, Leo Gallas b, Kamil Kovarck c,Bretislav Koudela a,d</p><p>aDepartment of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences Brno, Palackho tr. 1/3, 612 42 Brno, Czech RepublicbDepartment of Meat Hygiene and Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackho tr. 1/3, 612 42 Brno, Czech RepubliccDepartment of Virology and Diagnostics, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech RepublicdCentral European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackho tr. 1/3, 612 42 Brno, Czech Republica r t i c l e i n f o</p><p>Article history:Received 13 June 2013Received in revised form14 October 2013Accepted 2 November 2013Available online 13 November 2013</p><p>Keywords:Toxoplasma gondiiSurvivalTissue cystVacuum packagingDry fermented sausage* Corresponding author. Tel.: 420 54156 2269; faxE-mail address: (H. Neumaye</p><p>0740-0020/$ e see front matter 2013 Elsevier Ltd. b s t r a c t</p><p>Ingestion of raw or undercooked meat is a potential source of human toxoplasmosis. The aim of thisstudy was to determine the viability of Toxoplasma gondii cysts in vacuum packed (VP) goat meat and indry fermented sausages (DFS), and evaluate certain physical and chemical parameters, like water activity(aw), pH value, content of salt, dry matter and fat. A portion of muscle tissue from experimentallyinfected animals was used for production of VP meat with or without addition of 2.5% curing salt, andstored at 4 C or at 20 C. Results of bioassay showed that, samples of vacuum packed Toxoplasmapositive meat without salt addition were alive after six weeks at 4 C. Incubation at 20 C supported theviability after 3 h, but not after 4 h. After 7 days in 2.5% of curing salt, samples of T. gondii VP goat meatwere still viable, but not after 14 days at 4 C. All the DFS samples were not positive for infective cystswhich mean that, they do not pose a risk of T. gondii transmission. These data suggest that vacuumpackaging increases the survival of T. gondii cysts.</p><p> 2013 Elsevier Ltd. All rights reserved.1. Introduction</p><p>Toxoplasma gondii is a heteroxenous coccidian parasite ofhumans and warm-blooded animals with a worldwide distributionthat can cause serious illness, being dangerously virulent inimmunocompromised individuals and congenitally infected chil-dren. The sexual part of the life cycle occurs in feline carnivores,which excrete the oocysts in their faeces. After a 2e5 day longsporulation process, the oocysts become infectious and can betransmitted to other hosts through inadvertent ingestion. Asexualmultiplication in the intermediate host leads to the formation oftissue cysts in muscle tissue and organs, which are infectious forhosts that consume them, including humans (Dubey, 2004).Whether tissue cysts are the actual source of infection is usually notknown, but there are several reports of T. gondii induced outbreaksthat have been associated with the consumption of raw meat inCanada, Korea, French Guiana and New Zealand (McDonald et al.,1990; Choi et al., 1997; Carme et al., 2002; Lake et al., 2002).: 420 541562266.rov).</p><p>All rights reserved.After ingestion of tissue cysts by the intermediate host brady-zoites transform to tachyzoites, multiply locally, and disseminate inthe body. In pregnant females there is even possible transfer to thefoetus through the placenta. In many studies, ingestion of inade-quately cooked meat was linked to T. gondii infection in pregnancy(Kapperud et al., 1996; Bobic et al., 1998; Cook et al., 2000). How-ever, while the consumption of raw or undercooked meat wasconsistently identified as a risk factor for toxoplasmosis in manystudies, its relative importance and the meat type varied amongdifferent countries (Cook et al., 2000).</p><p>Many serological studies have been performed in livestock,but this line of research does not provide a true assessment ofthe toxoplasmosis risk for humans as the packing and storage ofmeat also affects the viability of T. gondii tissue cysts. In severalstudies, the susceptibility of cysts to various physical traumassuch as heat treatment, freezing, gamma irradiation or high-pressure, was tested (Dubey et al., 1990; Kuticic andWikerhauser, 1996; Lindsay et al., 2006). Heat treatment is themost secure way to inactivate tissue cysts while bradyzoites arealso destroyed by salting, curing, pickling and the use ofenhancing solutions, like sodium chloride, potassium lactate,sodium lactate etc., which can be injected in to ensure longerstability or better taste of final meat product, but some of these</p><p>Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:hneu.vfu@gmail.com;</p></li><li><p>Vacuum packed goat meat 50 g </p><p>Pepsin digestion</p><p>Bioassay in to 4 mice</p><p>Euthanasia after 2 months</p><p>SeraELISA</p><p>BrainPCR</p><p>Evaluation of T. gondii viability</p><p>Evalutation of T. gondii presence (Real-time PCR)</p><p>Fig. 1. Testing scheme for vacuum packed goat meat.</p><p>H. Neumayerov et al. / Food Microbiology 39 (2014) 47e5248treatments have not been standardized (Kotula et al., 1991;Lundn and Uggla, 1992; Hill et al., 2006). Vacuum packagingof meat is commonly used to preserve meat cuts and until nowthere have been no studies accessing the viability of T. gondiiafter the vacuuming process, which facilitates the longer shelf-life of the meat. In addition, dry fermented meat products havealso been implicated in the epidemiology of toxoplasmosis,because there is no thermal treatment step during thismanufacturing process. The production technology of dry fer-mented meat products is unique because of the number ofbiochemical processes that takes place during the ripeningphase, which contributes to the preservation of the final product.</p><p>Among the various kinds of commonly consumed meat, goatproducts pose a risk of infection due to their high susceptibilityto T. gondii, as exemplified by its high prevalence which rangesup to 77% of the meat on the market in some countries (Dubeyand Beattie, 1988; Tenter et al., 2000; EFSA, 2007; Dubey et al.,2011; Hill and Dubey, 2013). The assessment of risk depends notonly on the presence of antibodies against T. gondii but also onthe parasite quantity in meat. Although some studies have re-ported the isolation of T. gondii from caprine tissues and theirconsequent genotyping (Dubey et al., 2011), the level of infec-tion was not quantified. Recently, Jurnkov et al. (2013)described the distribution of T. gondii parasites in meat cutsand organs of experimentally infected, post-weaned goat kids.All tested muscles from shoulder, loin and leg, were found to bepositive for the parasite, and the highest parasite load was in thedorsal muscle tissue of goats euthanized 90 dpi. This studydirectly builds on this previous work. Its main aim was to accessthe viability of T. gondii cysts in VP1 meat from experimentallyinfected goats stored at different conditions and after themanufacturing of DFS2. Because past studies have lackeddetailed meat product characteristics, another goal was to testthe physical and chemical parameters like water activity, theactivity of the hydrogen ion (pH), content of salt, dry matter andfat of VP goat meat and DFS to define their influence on T. gondiibradyzoites survival.Dry fermented sausage 50 g </p><p>Pepsin digestionEvalutation of T. gondii </p><p>presence conventional PCR 2. Materials and methods</p><p>2.1. Experimental design</p><p>After experimental infection of naive goat kids, we obtainedfrom them meat containing T. gondii bradyzoites. Infected goatmeat was either processed by vacuum packaging, with or withoutcuring salt addition, or made into fermented sausages. All thesamples after these different treatment and storage conditionswere used in bioassays of four outbred mice to determine T. gondiicyst viability. The testing schemes of vacuum packed meat and dryfermented sausages are shown in Figs. 1 and 2, respectively.Bioassay in to 4 mice</p><p>Euthanasia after 2 months</p><p>SeraELISA</p><p>BrainPCR</p><p>Evaluation of T. gondii viability</p><p>from digested solution</p><p>Fig. 2. Testing scheme for dry fermented sausages.2.2. Experimental infection</p><p>Meat from sixteen shorthaired male post-weaned 64e68 dayold kids, which were experimentally infected with a suspension of20,000 oocysts of T. gondii of genotype II, Apico (Jurnkov et al.,2013) was used in this study. Animals were housed in the animalcare facility at the Ruminants and Swine Clinic of the University ofVeterinary and Pharmaceutical Sciences Brno and were handledwith the agreement of the Ethical Commission. The euthanasia wasperformed after intravenous injection of thiopental and cuttingjugular veins after passing out. The meat cuts were collected andconfirmed as T. gondii positive. The experimental infection of goatsis described in detail in Jurnkov et al. (2013).2.3. Production of vacuum packed goat meat</p><p>Infected meat from shoulders, loins and legs were collected andcut into small pieces of about 1 1 cm in size. Fifty grams of thesecubeswere individually packaged into bags. These bags consisted ofa 60 mm coat of polyamide (PA), 20 mm coat of polyethylene (PE)with an ethylene-vinyl acetate (EVA) oxygen barrier layer (AMILENPA/PE, Verpackungen GmbH, Germany). The manufacturer declaresthis barrier having a gas transmission rate of 50, 10 and150 cm3 m2 d1 permeability for O2, N2 and CO2, respectively, at23 C, 0% relative humidity. According to manufacturer, the watervapour transmission rate was 3.0 g m2 d1 at 23 C, 85% relativehumidity. Weight per area was 80 g m2, tensile strength at break45% longitudinally and 35% transversally. After being filled withmeat samples the 99.9% of the air was evacuated from the package,sealed using of a Vac-Star vacuuming packagingmachine (S 223 GX,Frimark CZ Ltd., Czech Republic). Vacuumed packages of goat meatwith the addition of 2.5% curing salt (sodium nitrite 6% and sodiumchloride 94%) or unsalted were stored at 2 2 C and stored for aperiods of 2 h as well as 7, 14, 21, 36 and 42 days. Other packages ofunsalted meat were stored at 20 C for 30 min as well as 1, 2, 3, 4,5 and 6 h. The temperature of stored meat (2 2 C) was</p></li><li><p>Table 1T. gondii survival in vacuum packed non-salted meat stored at 4 C according tobioassay in mice and ELISA and PCR based assays. Physical and chemical parametersof the infected meat are listed.</p><p>Time period Results of bioassayin ELISA/PCRa</p><p>pH SDb aw SDb</p><p>2 h (4/4)/(4/4) 5.541 0.002 0.993 0.0017 days (4/4)/(4/4) 5.743 0.005 0.973 0.00114 days (4/4)/(4/4) 5.775 0.010 0.974 0.00221 days (4/4)/(2/4) 5.747 0.008 0.974 0.00128 days (4/4)/(4/4) 5.813 0.002 0.976 0.00136 days (4/4)/(3/4) 5.875 0.012 0.976 0.00042 days (4/4)/(4/4) 6.015 0.010 0.975 0.000a Results of PCR and ELISA are given as (number of positive animals/number of</p><p>infected animals).b SD e standard deviation.</p><p>H. Neumayerov et al. / Food Microbiology 39 (2014) 47e52 49determined with a digital thermometer (GTH 175/Pt-E, Greisingerelectronic, Germany). All the samples were subsequently bio-assayed to mice after pepsin digestion.</p><p>2.4. Production of dry fermented goat sausages</p><p>Infected goat meat from loins, shoulders and legs was minced. Acommercial mixture of spices with saccharides for dry fermentedsausages (Maso-Profit, Czech Republic), starting culture (Chr.Hansen, Denmark), according to recommendations of manufac-turer, and 2.5% curing salt (sodium nitrite 6% and sodium chloride9%) were added to the sausage mixture during the grinding phase.The mixture was filled into 55 mm diameter collagen casings.Fermentation and ripening occurred in an air-conditioned chamber(Maurer, Germany); the initial temperature of 24 C was reducedgradually to 15 C. The relative humidity of the air was set at 94%,before being reduced to &lt; 80% after one week. Samples werecollected after 3 h as well as 1, 2, 3, 5 and 12 days. A bioassay using 4mice was conducted from material from each sausage after pepsindigestion.</p><p>2.5. Pepsin digest mediated isolation T. gondii bradyzoites</p><p>Each sample of meat or sausage was digested according to apreviously described modified digestion technique (Dubey, 1998)and bioassayed to 4 ICR mice. Mice were euthanized 2 months afterinfection and examined for the presence of T. gondii DNA in brainswith using PCR and T. gondii IgG antibodies in mice sera usingELISA.</p><p>2.6. Isolation of DNA from pepsin digested sausages</p><p>An approximately 250 ml aliquot from the digested sausagesamples was used for DNA isolation using the Nucleo Spin Tissue Kit(Macherey Nagel) according to the manufacturers protocol. TheDNA was eventually resuspended in 50 ml of PCR water. Sampleswere subsequently assayed by PCR to confirm the presence ofT. gondii using the program described in Section 2.7.</p><p>2.7. Collection of samples from bioassayed mice and DNA isolationfrom mice brains</p><p>After the euthanasia 2 months after infection, the mice brainswere removed. First the whole brainwas homogenized, then 25mgof tissue was lysed with 30 ml proteinase K treatment at 56 C. DNAfrom mouse brain was isolated with using the Macherey NagelTissue Kit according to manufacturers instructions and the DNAwas resuspended in 50 ml of PCR water. The animals were also bledand sera were obtained by centrifugation for 10 min at 0.4 rcf.</p><p>2.8. T. gondii detection by PCR</p><p>Primers TM1 and TM2 for the B1 gene were used for T. gondiidetection as previously described (Pujol-Riqu et al., 1999). TheT. gondii sequencewas amplified in 25 ml of reactionmixture consistof 3 ml DNA, 10 pmol of each primer, 7.5 ml PCR H2O and 12.5 ml PCRCombi PPPMaster Mix (150mM TriseHCl, 40mM (NH4)2SO4, 0.02%Tween 20.5 mM MgCl2, 400 mM dATP, 400 mM dCTP, 400 mM dGTP,400 mM dTTP, 100 U/ml Taq Purple DNA polymerase, 10 mM BSA TopBio, Czech Republic). The PCR programwith an initial denaturation(95 C for 3 min) was followed by 30 cycles of denaturation (94 C,0.5 min), annealing (65 C, 0.5 min) and extension (72 C, 1 min),plus a final extension run at 72 C for 10 min. Electrophoresis of thePCR product was performed on a 1.5% agarose gel and stained withGold View (Ecoli, The Slovak Republic); results were visualizedusing a UV illuminator (Vilbert Roumant, France).</p><p>2.9. Mice ELISA</p><p>The used ELISA preparation protocol for the detection of mouseIgG antibodies against T. gondii antigens was essentially asdescribed by Jurnkov et al. (2013). All serawere diluted 1:20 withs...</p></li></ul>