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Food Microbiology 39 (2014) 47e52

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Food Microbiology

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

Helena Neumayerová a,*, Jana Juránková a, Alena Saláková b, Leo Gallas b, Kamil Kova�r�cík c,B�retislav Koudela a,d

aDepartment of Pathology and Parasitology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého t�r. 1/3, 612 42 Brno, Czech RepublicbDepartment of Meat Hygiene and Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého t�r. 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, Palackého t�r. 1/3, 612 42 Brno, Czech Republic

a 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.: þ420 54156 2269; faxE-mail address: hneu.vfu@gmail.com (H. Neumaye

0740-0020/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.fm.2013.11.001

a 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).

: þ420 541562266.rová).

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; Bobi�c 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

Vacuum packed goat meat 50 g

Pepsin digestion

Bioassay in to 4 mice

Euthanasia after 2 months

SeraELISA

BrainPCR

Evaluation of T. gondii viability

Evalutation of T. gondii presence (Real-time PCR)

Fig. 1. Testing scheme for vacuum packed goat meat.

H. Neumayerová et al. / Food Microbiology 39 (2014) 47e5248

treatments have not been standardized (Kotula et al., 1991;Lundén 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, Juránková 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

SeraELISA

BrainPCR

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 (Juránková 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 Juránková 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)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 m�2 d�1 permeability for O2, N2 and CO2, respectively, at23 �C, 0% relative humidity. According to manufacturer, the watervapour transmission rate was 3.0 g m�2 d�1 at 23 �C, 85% relativehumidity. Weight per area was 80 g m�2, 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

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.

Time period Results of bioassayin ELISA/PCRa

pH � SDb aw � SDb

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 � 0.000

a Results of PCR and ELISA are given as (number of positive animals/number ofinfected animals).

b SD e standard deviation.

H. Neumayerová et al. / Food Microbiology 39 (2014) 47e52 49

determined with a digital thermometer (GTH 175/Pt-E, Greisingerelectronic, Germany). All the samples were subsequently bio-assayed to mice after pepsin digestion.

2.4. Production of dry fermented goat sausages

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 < 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.

2.5. Pepsin digest mediated isolation T. gondii bradyzoites

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.

2.6. Isolation of DNA from pepsin digested sausages

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 manufacturer’s 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.

2.7. Collection of samples from bioassayed mice and DNA isolationfrom mice brains

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 manufacturer’s 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.

2.8. T. gondii detection by PCR

Primers TM1 and TM2 for the B1 gene were used for T. gondiidetection as previously described (Pujol-Riqué 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 with

Gold View (Ecoli, The Slovak Republic); results were visualizedusing a UV illuminator (Vilbert Roumant, France).

2.9. Mice ELISA

The used ELISA preparation protocol for the detection of mouseIgG antibodies against T. gondii antigens was essentially asdescribed by Juránková et al. (2013). All serawere diluted 1:20 withsample dilution buffer (PBS-T with 2.5% casein hydrolysate) andincubated for 1 h in a wet chamber at 37 �C. The wells were washedthree times in PBS-T and 100 ml peroxidase-conjugated Goat Anti-Mouse IgG (H þ L, Jackson ImmunoResearch) was added into eachwell. After incubation (30 min, 37 �C) and washing, 100 ml of TMBComplete substrate-chromogen solution (Test-line, Brno, CZ) wasadded into each well. The reaction was stopped after 15 min ofincubation by adding 100 ml of 2M sulphuric acid and absorbancewas registered at 450 nm using the multichannel spectrometeriEMS Reader (Labsystems, Helsinki, Finland). Negative controlswere obtained from sera of SPF outbred mice. Positive control wasobtained from SPF outbred mice experimentally infected with 1000sporulated oocysts of T. gondii tiger isolate of genotype II, Apico I(Juránková et al., 2013). The borderline of positive value wasestablished based on the examination of serum samples obtainedfrom 30 SPF rabbits (Charles River Laboratories Germany, Munich,Germany). For the mice T. gondii IgG antibodies the borderline forwas the borderline established for 0.40 of OD of positive control.

2.10. Chemical and physical parameters

The following chemical and physical parameters of themeat andsausages were also determined. The amount of dry matter wastested according to ISO 1442:1997 (Meat and Meat Products:Determination of Moisture Content Reference Method). Fat wasanalysed on a Soxtec analyser (FOSS Tecator, Sweden) with diethylether as the extraction agent (Application Sub Note 3127, 2001).Value of pH was measured with a SenTix Sp needle probe attachedto a 340iWTWpH-meter (WTW,Weilheim, Germany). The amountof chloride salts was determined using the Volhard method (CSNISO 1841-1). Water activity (aw) was measured on Novasina Lab-Master analyser (Novasina, Switzerland) at 25 �C.

3. Results

Vacuum packed non-salted meat was stored at 4 �C for 2 h aswell as 7, 14, 21, 28, 36 and 42 days. Tissue cysts of T. gondii survivedin all samples of non-salted meat as revealed by bioassay of eachsample from four mice. The detailed results of the ELISA test andPCR are depicted together with the sourcemeat aw and pH values inTable 1. Non-salted vacuum packed goat meat was also exposed to

Table 3T. gondii survival in vacuum packed salted meat stored at 4 �C as determined bybioassay in mice as well as ELISA and PCR based assays. Physical and chemical pa-rameters of the infected meat are listed.

Time period Results ofbioassay inELISA/PCRa

pH � SDb aw � SDb Content ofsalt % � SDb

2 h (4/4)/(4/4) 5.704 � 0.008 0.977 � 0.000 1.90 � 0.027 days (4/4)/(3/4) 5.541 � 0.006 0.961 � 0.001 1.90 � 0.2014 days (0/0)/(0/0) 5.825 � 0.008 0.960 � 0.002 1.86 � 0.7021 days (0/0)/(0/0) 5.825 � 0.006 0.967 � 0.001 1.92 � 0.1028 days (0/0)/(0/0) 5.872 � 0.012 0.969 � 0.000 1.91 � 0.0136 days (0/0)/(0/0) 5.895 � 0.006 0.971 � 0.001 1.97 � 0.1942 days (0/0)/(0/0) 5.975 � 0.005 0.966 � 0.002 1.98 � 0.5

a Results of PCR and ELISA are given as (number of positive animals/number ofinfected animals).

b SD e standard deviation.

H. Neumayerová et al. / Food Microbiology 39 (2014) 47e5250

freezing conditions at �20 �C. The bioassay results suggest thatT. gondii cysts were still vital after 3 h under these conditions butlost viability after 4 h; detailed results are shown in Table 2.

The viability of T. gondii VP salted meat was documented after 7but, not after 14 days. The physical and chemical parameters of theinfected meat serve as other variables that can influence parasitesurvival: lethal meat aw was determined as 0.960 � 0.002,1.856% � 0.70% salt content and 5.825 � 0.008 pH. The meatphysical and chemical parameters along with their correspondingbioassay results for vacuum packed salted goat meat samples areshown in Table 3.

T. gondii cysts were nonviable in all samples of dry fermentedsausages using bioassay in mice. The T. gondii DNA was detected inall samples of sausages. The important chemical and physical pa-rameters for dry fermented products are described in Table 4 alongwith results from assays for parasite presence in the testedsausages.

4. Discussion

The importance of food safety has been growing in recently.Although toxoplasmosis is the most reported parasitic zoonosis inhumans, the importance and frequency of T. gondii infection viagoat meat and goat meat products remains unknown in the EU(EFSA, 2007).

Furthermore, the viability T. gondii in (VP) meat has still notbeen tested until now. The results in this study can only becompared with data from other studies on conventionally treatedmeat, where the process of spoilage is a limiting factor, or studieson isolated tissue cysts. Survival in of isolated tissue cysts in buff-ered saline stored at 4 �Cwas long ago documented for up 2months(Jacobs et al., 1960) and even some tissue cysts survived for severaldays after the death of an infected animal, although though itstissues have begun decomposing (Work et al., 2000). But if theT. gondii positive meat is fresh and it is not thermally treatedwe canbe sure, that cysts will be infectious, theoretically we can concludethat if the VP meat maintain its freshness T. gondii cysts can survivefor longer time than 6 weeks in this meat.

Treatments of meat with various enhancing solutions and saltare commonly used in meat industry (Dubey et al., 2005). Dubey(1997) tested isolated tissue cysts in 6% NaCl solution at tempera-tures from 4 to 20 �C. At 4 �C, isolated tissue cysts survived for atleast 56 days in 0.85% NaCl, for 49 days in 2.0% NaCl, and 21 days in3.3% NaCl. In our study, the vacuum packed salted meat with1.86 � 0.70% salt content was infectious for 7 but not for 14 days at4 �C, a much shorter period than that for the isolated tissue cysts.Our results thus suggest that from a consumer food safety point ofview, it is better to consume prefabricated vacuum packed meatwith addition of salt and spice because the chance of infectioncould be lower. From the work of Dubey (1997) it was documentedthat tissue cysts are killed with treatment of conventional salt. Thestudy by Hill et al. (2004) demonstrated that injection of mousebrains or pork loins with solutions containing 2% sodium chloride

Table 2T. gondii survival after meat freezing at �20 �C as assayed by ELISA and PCR.

Time period Results of bioassay in ELISA/PCRa

30 min (4/4)/(4/4)1 h (4/4)/(3/4)2 h (4/4)/(4/4)3 h (3/4)/(2/4)4 h (0/0)/(0/0)5 h (0/0)/(0/0)6 h (0/0)/(0/0)

a Results of PCR and ELISA are given as (number of positive animals/numberof infected animals).

or 1.4% potassium or sodium lactate, alone or in combination withother components, prevented transmission of T. gondii to cats. Inthe next study by Hill et al. (2006), enhancement solutions withvarious concentrations of sodium chloride, sodium diacetate, so-dium tripolyphosphate, potassium lactate or sodium lactate wasused as injection to T. gondii positive meat. Results indicate that, thetissue cysts within 8 h of exposure are devitalized (inactivated)after 8 h with exception of 0.85% NaCl with combination of 0.2%sodium tripolyphosphate treatment, where the viability wasconfirmed after 40 h. If we compare our results of VP salted meat inour study we can conclude that 2.5% initial amount of sodium ni-trite was effective in killing of T. gondii cysts in 14 days, which ismuch later than in previously mentioned studies. Also otherenhancement solutions should be tested in vacuum packagingconditions, because the survival of tissue cysts could be prolongedby this procedure even in the case of addition of variousenhancement solutions.

Perhaps a more effective method to prevent T. gondii trans-mission is by storage at freezing temperatures. While this strategyis widely used by consumers, it is also generally discouraged due toits negative impact on taste. However, this method ultimatelycontributes to food safety as T. gondii tissue cysts are quicklydevitalized. (Sommer et al., 1965; Grossklaus and Baumgarten,1968; Dubey, 1988; Kuticic and Wikerhauser, 1996; Djurkovic-Djakovic and Milenkovic, 2000). Similar results were obtained byDubey (1974) where T. gondii was devitalized after 3 or more hoursat �20 �C in whole frozen mice, portions of sheep brains or sheepmuscles. But if the temperatures range around �1 �C and �3.9 �Cthe cysts remained infectious even after 22 days (Kotula et al.,1991). These findings should be bear in mind to prevent theinfection and the temperature of the freezers can be checked toestimate the survival rate of T. gondii in meat.

In our study, the meat portion may be small, weighing 50 g.Thus, it is necessary to define the packaging size because parasitesurvival in bigger portions may actually be better.

Dry fermented sausages represent major products in the meatprocessing industry. T. gondii potential survival in these types ofproducts during the ripening process is not well known. All thesamples of dry fermented sausages in our study were positive inPCR for T. gondii DNA, thus the parasite was present in all testedsamples. But the bioassay did not reveal any positive result. Thereare some studies describing T. gondii survival in processed meatproducts. In swine sausages from Chilean commercial establish-ments, almost half of the samples (47.14%) were positive forT. gondii DNA, but were negative in the infection bioassay (deOliveira Mendonca et al., 2004). In contrast, viable parasites weredetected in one out of 67 cured meat samples investigated in theUnited Kingdom (Warnekulasuriya et al., 1998). It has been also

Table 4T. gondii survival in dry fermented sausages as assayed by ELISA and PCR. Physical and chemical parameters of the infected meat are listed.

Maturation period Results of bioassay in ELISA/PCRa pH � SDb aw � SDb Content of salt (%)� SDb Content of fat (%)� SDb Content of dry matter (%)� SDb

2 h (0/4)/(0/4) 5.602 � 0.003 0.964 � 0.002 1.76 � 0.10 6.55 � 1.30 31.85 � 0.581 day (0/4)/(0/4) 5.100 � 0.014 0.955 � 0.001 2.38 � 0.11 7.98 � 0.85 32.97 � 0.402 days (0/4)/(0/4) 5.039 � 0.010 0.950 � 0.003 2.83 � 0.11 10.52 � 1.01 36.94 � 0.303 days (0/4)/(0/4) 5.016 � 0.004 0.942 � 0.002 3.21 � 0.14 14.00 � 1.20 42.22 � 0.325 days (0/4)/(0/4) 4.910 � 0.021 0.922 � 0.001 3.52 � 0.18 15.36 � 0.92 50.64 � 0.5112 days (0/4)/(0/4) 5.131 � 0.012 0.814 � 0.005 4.55 � 0.11 17.24 � 0.89 69.65 � 0.78

a Results of PCR and ELISA are given as (number of positive animals/number of infected animals).b SD e standard deviation.

H. Neumayerová et al. / Food Microbiology 39 (2014) 47e52 51

shown that salting does not necessarily kill tissue cysts in otherhome-made pork sausages (Jamra et al., 1991; Navarro et al., 1992).The presence of viable T. gondii organisms in commercially madefresh pork sausages was recently shown in a study from Brazil (Diaset al., 2005). However, the lack of information about the sausagesalt concentration, period of curing and other commonly used pa-rameters in meat industry, such as water activity and pH, make thecomparison between these studies rather difficult.

Methodological approach to assess T. gondii viability inmeat andmeat products is different across before mentioned studies. In thework of Hill et al. (2006), seronegative cats were fed with infectedsamples to assess T. gondii viability. However, this approach is nownot possible, and thus the described modified digestion techniqueprocessing infected meat and the subsequent bioassay in mice, aspresented in our study, serves as a proxy for parasite transmission.The advantage of this method is requirement for a big initialamount of sample (50 g). For the final assessment of T. gondii sur-vival PCR and ELISA T. gondii detection assays were used, therewereinternal inconsistencies between the results of both methods.Detection of T. gondii IgG antibodies from mice sera was found outto be more specific than PCR using DNA extracted from brainapparently due to a small part of the used organ, which could leadto false negatives as the distribution of the parasite is irregular.

In our study, we revealed that T. gondii bradyzoites lost infec-tivity in all samples from dry fermented sausages, even though theused goat meat was positively tested for T. gondii by bioassay andPCR positive. Apparently, this products’s high salt content and lowwater activity is not amenable for T. gondii bradyzoite survival. Thusfermented meat products can be considered to be safe for con-sumption by high- risk individuals like immunosuppressed peopleand pregnant women.

We have performed an initial study on T. gondii viability invacuum packed goat meat and dry fermented goat sausages. Ourstudy has shown that T. gondii was not viable in frozen vacuumpacked meat and fermented sausages. Furthermore, we describemeat physical and chemical parameters that appear to be detri-mental to T. gondii tissue cysts. We suppose that, vacuum packingsignificantly contributed to T. gondii cyst survival when non-saltedgoat meat was used. Thus, from a public health standpoint, it issafer to consume vacuum packed meat stored at temperaturesabove freezing that has been supplemented with salt. We can alsoconclude that there is urgent need for further development andstandardization of methods for the testing of the pathogen survivalin various meat products.

Acknowledgements

This work was supported by the Ministry of Sports and Educationof the Czech Republic (Grant No. MSM6215712402) and by the “CEI-TEC e Central European Institute of Technology” project (CZ.1.05/1.100/02.0068) from the European Regional Development Fund.

We also thank the personnel at the Ruminant and Swine Clinicof the University of Veterinary and Pharmaceutical Sciences in Brno

for housing experimental animals and help with sampling duringthe study.

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