survival of experimentally induced toxoplasma gondii tissue cysts in vacuum packed goat meat and dry...
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Food Microbiology 39 (2014) 47e52Contents lists avaiFood Microbiology
journal homepage: www.elsevier .com/locate/ fmSurvival of experimentally induced Toxoplasma gondii tissue cysts invacuum packed goat meat and dry fermented goat meat sausages
Helena Neumayerov a,*, Jana Jurnkov a, Alena Salkov b, Leo Gallas b, Kamil Kovarck c,Bretislav Koudela a,d
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
Article history:Received 13 June 2013Received in revised form14 October 2013Accepted 2 November 2013Available online 13 November 2013
Keywords:Toxoplasma gondiiSurvivalTissue cystVacuum packagingDry fermented sausage* Corresponding author. Tel.: 420 54156 2269; faxE-mail address: firstname.lastname@example.org (H. Neumaye
0740-0020/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.fm.2013.11.001a b s t r a c t
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.
2013 Elsevier Ltd. All rights reserved.1. Introduction
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).
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).
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
Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:email@example.com://crossmark.crossref.org/dialog/?doi=10.1016/j.fm.2013.11.001&domain=pdfwww.sciencedirect.com/science/journal/07400020http://www.elsevier.com/locate/fmhttp://dx.doi.org/10.1016/j.fm.2013.11.001http://dx.doi.org/10.1016/j.fm.2013.11.001http://dx.doi.org/10.1016/j.fm.2013.11.001
Vacuum packed goat meat 50 g
Bioassay in to 4 mice
Euthanasia after 2 months
Evaluation of T. gondii viability
Evalutation of T. gondii presence (Real-time PCR)
Fig. 1. Testing scheme for vacuum packed goat meat.
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.
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
Pepsin digestionEvalutation of T. gondii
presence conventional PCR 2. Materials and methods
2.1. Experimental design
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
Euthanasia after 2 months
Evaluation of T. gondii viability
from digested solution
Fig. 2. Testing scheme for dry fermented sausages.2.2. Experimental infection
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
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