Quality Control of Toxoplasma Gondii in Meat Packages

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Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISAtest and its use for detection in rabbit meat cuts

Juliana Nunes Mecca, Luciana Regina Meireles, Heitor Franco de Andrade Jr.

Laboratrio de Protozoologia do Instituto de Medicina Tropical da Universidade de So Paulo, Av. Dr. Enas de Carvalho Aguiar, 470, 05403-000 So Paulo, SP, Brazil

a b s t r a c ta r t i c l e i n f o

Article history:

Received 25 June 2010

Received in revised form 18 January 2011Accepted 20 January 2011

Keywords:

Toxoplasmosis

Meat

Packages

Quality control

ELISA

Meat juice

Toxoplasma gondii causes severe disease both to manand livestock andits detection in meat after slaughtering

requires PCR or biological tests.Meat packages contain retained exudate that could be used forserology due to

its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-

T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by

cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or

meat juice, after blood content correction. These assays were adequate regardless of the storage time up to

120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in

commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat

juice may be useful for quality control for toxoplasmosis monitoring.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Toxoplasmosis is a widespread cosmopolitan zoonosis that iscaused by the obligatory intracellular protozoan, Toxoplasma gondii,

an agent that infects warm-blooded animals, including humans

(Dubey & Beattie, 1988). It is estimated that one third of the global

human population has been infected with this parasite (Tenter et al.,

2000). The infection is generally asymptomatic or associated with

mild, non-specific, acute clinical symptoms (Remington et al., 1995).

Congenital toxoplasmosis generally occurs when the primary infec-

tion is acquired during pregnancy and may result in abortion or the

re-appearance of symptoms later in life, including ocular toxoplas-

mosis, which may also occur in infections acquired after birth

(Remington et al., 2006). In immune-suppressed individuals, the

disease may lead to lethal encephalitis or systemic disease (Luft &

Remington, 1992). The disease is acquired by ingestion of vegetables

and water containing oocysts from feline stools, through vertical

transmission during pregnancy, or by ingestion of raw or poorly

cooked meat containing cysts (Hill & Dubey, 2002). T. gondii was

responsible for 21% of deaths attributed to pathogens transmitted by

food in the United States (Mead et al., 1999).

Small outbreaks of toxoplasmosis have been associated with the

consumption of raw meat in Korea, USA, France, French Guiana and

New Zealand, (Kijlstra & Jongert, 2008). Most farm animals that are

naturally infected by T. gondii have been shown to carry infectious

parasites in their meat, with the possible but questionable exception

of beef (Zia-Ali et al., 2007). Ground or minced meat could becontaminated by other types of meat during the grinding process,

which could explain the epidemiological linkage between the

consumption of raw beef and small outbreaks of infection by T. gondii

(Dubey & Jones,2008). There are several new ready-to eat smallgoods

which are meat products that may represent a source of T. gondii

infection, as elsewhere discussed (Mie et al., 2008).

Despite efforts in individual countries or in individual industries,

there is no sanitary control or laboratory certification program related

to screening for T. gondii cysts in meat destined for human

consumption (Kijlstra & Jongert, 2009). Bioassay and molecular

biology tests used to search for cysts in meat are complex and time

consuming due to the uneven distribution of cysts in the carcass,

which often results in false negatives(Lundnet al., 2002). Antibodies

usually reflect the contact of the host with the parasite and could also

reflect the infective status of meat (Dubey & Jones, 2008). ELISA is a

large scale, simple and sensitive serological assay method that is useful

for the surveillance and control of toxoplasmosis (Ferguson et al.,

1989). This diagnostic test has been conducted for sanitary control

using serum samples taken from slaughtered animals, despite few

sampling associated problems. This preventive approach is safe and

useful, but frequently the only available sample for testing is already

commercialized meat,which contains exudates,formed after the retail

processing, and mainly composed of blood and interstitial fluid. As

reported elsewhere, there is a good correlation between ELISA results

obtained for anti-T. gondii antibodies detected from meat juices and

from serum samples (Lundn et al., 2002; Wingstrand et al., 1997).

Meat Science 88 (2011) 584589

Corresponding author at: Av. Dr. Eneas de Carvalho Aguiar, 470, 05403-000, So

Paulo, SP, Brazil. Tel.: +55 11 30617010; fax: +55 11 30885237.

E-mail addresses: [email protected] (J.N. Mecca), [email protected]

(L.R. Meireles), [email protected] (H.F. de Andrade).

0309-1740/$ see front matter 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.meatsci.2011.01.016

Contents lists available at ScienceDirect

Meat Science

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i
http://dx.doi.org/10.1016/j.meatsci.2011.01.016http://dx.doi.org/10.1016/j.meatsci.2011.01.016http://dx.doi.org/10.1016/j.meatsci.2011.01.016mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.meatsci.2011.01.016http://www.sciencedirect.com/science/journal/03091740http://www.sciencedirect.com/science/journal/03091740http://dx.doi.org/10.1016/j.meatsci.2011.01.016mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.meatsci.2011.01.016


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Same correlations were also observed in ELISA assays used to detect

antibodies in othermeat-transmitted infectious diseases(Davieset al.,

2003; De Lange et al., 2003; Kapel et al., 1998). However, most of the

authors agree that the main problem with these particular tests is due

to therate of false negative samples causedby lowbloodlevels in meat

juices, resulting in an approval of infective meat products as clean and

safe for consumption.

The blood content could be determined by the spectrophotometry

of cyanhemoglobin in a microplate ELISA reader, as elsewhere des-cribed (Frenchik et al., 2004). Rabbits are produced for meat con-

sumption in Brazil and are common experimental animals with

colony facilities in our institution (Tavares et al., 2007), thus providing

a model for quality control. We performed anti-T. gondii ELISA on

meat juices of packaged muscles from experimentally infected rabbits,

adjusting for blood content, looking for antibody specificity in

immunoblotting and also the stability of muscle juice after several

freezing and thawing process. Assays to detect infection of Brazillian

rabbit meat were also performed.

2. Materials and methods

2.1. Animals

For the standardization of an ELISA detection system, we used

recently weaned rabbits (New Zealand strain) showing a negative

serology for T. gondii (determined by ELISA) prior to experimental

inoculation. The animals were obtained from the animal colony of the

School of Medicine at Sao Paulo University (FMUSP) and were housed

at the Institute of Tropical Medicine of Sao Paulo (IMT-USP), receiving

commercial food and water ad libitum. The rabbits were properly

anesthetized before all of the invasive procedures were conducted,

and all of the procedures were conducted according to The Principles

of Ethics in Animal Experimentation (COBEA Brazilian School of

Animal Experimentation). For detection of possible natural infection

in rabbit meat for human consumption, we obtained cuts during

slaughtering of Botucatu strain rabbits, a lineage derived from New

Zealand rabbits with crosses of Norfolk strain devoted to meat

production (Moura et al., 2001). Rabbits were fed with a commercialdiet andoccasionally with green grass,withfiltered waterat libitum in

individual suspension cages at the production site until the time of

slaughtering at the abattoir. Animals were 6 months old and weighed

at least 5 kg.We collected individual serum samples andsquare pieces

of 10 g from anterior abdominal muscle in plastic bags from 74

rabbits, both immediately frozen and stored at 17 C until use.

2.2. Parasites

Tachyzoites of the RH strain of T. gondii were maintained by

cryopreservation in liquid nitrogen. The tachyzoites for antigen

production were recovered using successive intraperitoneal (i.p.)

passages in Swiss mice, at the Laboratory of Protozoology at IMT-USP.

The cystogenic ME49 strain ofT. gondii for experimental infection wasmaintained by successive oral passages of brain cysts from infected

C57Bl/6j mice.

2.3. Experimental infection of rabbits and sampling

Rabbits in the infection group (n= 4) were subcutaneously

inoculated with 10 tissue cysts of ME49 strain of T. gondii, while the

control group (n=3) was inoculated subcutaneously with 1 ml of

sterile saline solution. Both groups remained under the same

conditionsthroughout the study. Infectionprogression was monitored

by serology evolution in biweekly blood collection for 90 days, when

euthanasia was performed by usual anesthesia followed by cardiac

complete bleeding with serum sampling. Brain, liver and heart were

submitted to routine histology. Whole blood samples collected from

cardiac bleedingwere also distributed in sterile 1.5 ml micro tubesand

stored frozen at17 C, for freezingand defrosting similarlyto muscle

to obtain a 100% blood frozen control. Random 50 g cuts of meat were

obtained, packed into sealed plastic bags and immediately frozen at

17 C. Frozen meats were thawed overnightat room temperature to

obtain meat juices samples that were collected from the plastic bags.

Several cuts from the same animals remained stored at 17 C and

were thawed between 060 days forearlydefrostingand 60120 days

for longer defrosting and replicated three times per period to evaluatethe stability of antibodies in meat juices. After first meat juice collec-

tion, thawedcuts hadtheir plastic bagresealed andrefrozen at17 C

for at least 5 days Refrozen meat samples were submitted to a second

thawing formeat juice sampling to evaluate thepresenceand stability

of antibodies after successive thawing of the same meat sample.

2.4. Determination of hemoglobin concentration

After thawing, defrosted blood and meat juice samples were

diluted (1:10 v/v) in buffer Tris/HCl 10 mM pH 7.5 NH4Cl 150 mM,

NaN3 for erythrocyte lysis and conversion of hemoglobin to stable

cyanhemoglobin (Frenchik et al., 2004). Diluted samples were

centrifuged for 5 min at 12,000 g and supernatants were stored at

4 C. Previously, assuming a concentration of 15 g Hb/100 ml of blood,

processedblood was seriallydiluted and theA540nm were estimated in

a microplate reader (100 l/well), allowing a construction of a linear

regression model, with r2=0.9433 and540nm= 17.76, highly directly

correlated.

2.5. ELISA

Immunodetection was performed as previously described (Meireles

et al., 2004). RH strain T. gondii tachyzoite saline antigen was used as

immunosorbent.Briefly, diluted serum (1/100), freeze thawblood (1/10)

and meat juice (1/10) were cleared by centrifugation for 5 min at

12,000 g. Duplicates (100 ul) were serially diluted and applied in 96 well

microplate. The blood content was estimated by determination of A540nmin an automatic microplate reader, according to the method described

above (part 2.4). Alternatively, meat juices samples were diluted inanother microplate in order to dispense 0.03 A540nm per well,

corresponding to a blood quantity equivalent to a 1/100 blood dilution.

The use of a microplate and the A540nm results are feasible from mass

testing, using only one more step for technician or in the liquid handling

robot. Sampleswere transferred to antigencoated 96 well microplate and

incubated at 37 C for 1 h, with subsequent reaction to peroxidase anti

rabbit IgG conjugate and O-phenilenediamine peroxidase substrate, as

elsewhere described (Meireles et al., 2004). Results were expressed as

A492nm.

2.6. Immunoblotting of IgG from meat juices

Saline soluble tachyzoite protein from tachyzoites ofT. gondii were

subjected to SDS-PAGE (Laemli, 1970), in a gradient 5-20% SDS PAGEsystem. After electrophoresis, proteins were transferred from gel to a

nitrocellulose membrane, using semi-dry electrophoresis in 25 mM

Tris, 19 2 mM glycine, 205 methanol, pH 8.3, at constant current for

30 min. The membrane was removed, protein stained with 0.5%

Ponceau's staining solution, cut in 5 mm strips, and free binding sites

blocked by immersion in 20 ml of PBS 0.01 M pH 7.2, 0.05% Tween-20

(PBST)and 5%non fat dry milk, with constantgentle shaking for 2 h at

room temperature. Subsequent processes were conducted at room

temperature with five washes with PBST between steps. Strips were

individually reacted overnight at 4 C with sera (1:500) or with meat

juice, adjusted by the A540nm to the same dilution, in individual trays.

Strips were incubated for 1 h at room temperature with appropriate

dilution of anti rabbit IgG peroxidase conjugate. Bound conjugate was

revealed by Diaminobenzidine 1.0 mg/ml in citrate buffer 0.1 M pH

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6.0containing 0.01% H2O2. After staining, the reaction was interrupted

by washing in MilliQ water,and digital imaging obtained of each strip.

2.7. Stability of meat juices of animals experimentally infected with

T. gondii

The stability of meat juice samples was evaluated by comparing

ELISA results from exudates obtained after 1 or 2 thawing cycles at

different periods, as early (7

30 days) or longer (60

120 days) frozenperiods. These two approaches were used in association, resulting in a

larger number of meat samples that was adequate for comparing the

common storage meat conditions.

2.8. Meat juice ELISA from commercial rabbit meat samples

We analyzed the resultsof meat juices from anterior abdominal cuts

from 74 rabbits obtained during slaughtering in an abattoir that

provides rabbit meat to So Paulo area. Those animals were breed for

rabbit zootechnology purpose. Sera were obtained from 74 animals.

After one week of frozen storage at17 C, meat juice was individually

obtained as described above, adjusted to 0.03 A540nm and ELISA tested,

with results expressed as A492nm. Threshold values were established by

testing ELISA in meat juice of cuts from 25 seronegative samples, using

the mean plus 3 standard deviations.

2.9. Statistical analysis

Analysis offitting to sigmoidal dose-response curves with variable

slopeor linear regression models wereperformed usingmathematical

models in the software0 Prism 3.0. The estimated adjustment of r2

model and the probability of association of the Snedecor F were

evaluated. Serological indexes and confidence intervals were esti-

mated using EpiInfo 6.01 software.

3. Results

3.1. Experimental infection

T. gondii experimental infection in rabbits was evaluated by

detection of IgG antibodies using ELISA methods. Infected animals

showed a significant increase of antibodies in the second week after

inoculation with theagent, with a peak at 25 days post-infection. High

levels of antibodies were maintained until the end of the experiment.

Experimental infection was also confirmed by histopathology, with

presence of cysts in brain with smaller foci of encephalitis, liver portal

mononuclear cells infiltrate and small areas of the myocarditis (data

not shown).

3.2. Correlation of blood content in meat juice in anti T. gondii IgG ELISA

ELISA was performed to determine anti-T. gondii IgG presence

using serial dilutions of serum, blood and meat juices in experimen-tally infected rabbits samples. Results, shown in Fig. 1, were expressed

a s E LI SA A492nm, according to cyanhemoglobin A540nm of

corresponding dilutions, allowing excellent fitting to a sigmoidal

dose-response curve. Adjusting the blood content, samples of blood,

serum and meat juices give ELISA values that were quite similar,

regardless of the source. This finding demonstrated that detection of

anti-T.gondii IgG in meat juices had the same efficiency as in serum,

with good differentiation between positive and negative samples.

However, meat juice from non infected animals presented back-

ground interference in lower dilutions, higher than serum samples of

the same animals. The best discrimination between the sigmoidal

curves from samples from infected and non infected rabbits were

obtained around 0.03 A540nm in meat juice, representing a 1:200

serum dilution or 1:100 blood dilution.

3.3. Antigen specificity of IgG from several sources determined by

immunoblotting

The specificity of antibodies present in meat juiceswas analyzed in

immunoblotting analysis against T. gondii RH antigens, compared to

its serum counterpart, as described in Methods. Antigen recognized by

IgG in each sample was shown in Fig. 2. For each of the three infected

rabbits, the pattern observed after incubation with meat juice was

similar to that of serum. Absent or weak signal reactions were found

with T. gondii antigens when non-infected animal meat juices or

serum were tested, showing the specificity of ELISA and immuno-

blotting systems against specific anti-T.gondii IgG rabbits.

3.4. Quantitative data of anti-T. gondii IgG ELISA

Several meat samples were prepared from experimentally infected

or control rabbits andtheir meat juice resultedin 96 samples that were

tested in ELISA. Their results are shown in Fig. 3. The first approach by

conventional dilution did not allow an adequate distinction between

samples from infected animals and controls (Fig. 3), with 20 false

negative results and low sensitivity and negative predictive values

(Table 1). The second approach was performed by adjusting the blood

content in each meat juice before ELISA assay, 112 samples from

Fig. 1. ELISA anti-T. gondii IgG in serial dilution of serum, blood processed and meat

juice. X axis presented estimated blood content in each sample dilution, expressed as

LOG10 A540nm of cyanhemoglobin. Y Axis presented anti-T. gondii IgG ELISA as A492nm.

Error bars represents intra assay variation. Solid lines represent estimated sigmoidal

curves of each sample. Vertical line indicates the best discrimination of blood

concentration between positive and negative animals.

Fig. 2. Antigen specificity of meat juice and serum IgG against immunoblots of SDS-

PAGE ofT. gondii antigens. Lanes 1, 3, 5 and 7: meat juices. Lanes 2, 4, 6 and 8: serum.

Lane 1 and 2: Infected rabbit 1; lane 3 and 4: Infected rabbit 2; lane 5 and 6: Infected

rabbit 3. Lane 7 and 8: Control negative rabbit. Lane 9: Molecular weight markers.

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different meat juices were tested in one dilution, adjusted individually

forafixedand standardvalueof 0.03 A540nm,whichwasdefined during

the initial ELISA standardization as the best discrimination between

samples from infected or control animals. This approach allowed a

complete differentiation between samples from infected animals and

controls (Fig. 3), without any false negative results. This assay pre-

sented serological indexeswith higher efficiency, withhigh sensitivity,

specificity and positive and negative predictive values ( Table 1). This

test was performed at least three times with the same meat samples,

with good intratest and intertest reproducibility (r2=0.9972,

r2=0.9928, respectively, P valueb0.0001).

3.5. Meat juice stability after storage and defrosting

The stability of meat juices for the detection ofT. gondii was tested

after various freezing periods and also for at least one defrosting and

refreezing process of rabbit meat, as shown in Fig. 4. We tested

samples of meat juice samples from the first and second thawing of

the same sample of meat with similar results, with no degradation of

the antibodies when the meat is subjected to more than one cycle

of freezing and thawing. Meat juice from different pieces of meat

from the same animal also presented similar results, regardless of the

freezing time, with stability of the test for at least 120 days of frozen

storage.

3.6. Detection of anti-T. gondii IgG in meat juices from cuts of Brazilian

rabbits

We tested 74 samples from muscle from meat production rabbits,as described in methods. Meat juice from packaged cuts was obtained

by overnight defrosting and was immediately processed by ELISA

using theblood content correction for0.03 A540nm dilution. Results are

displayed in Fig. 5, indicating that only one animal was consideredpositive by meat juice ELISA, with a 1.35% occurrence of anti-T. gondii

IgG antibodies, showing the feasibility of this assay for Toxoplasma

infection detection in meat packages. The serum sample of the same

animal was also clearly positive. Results were reproduced in three

independent ELISA assays of the same meat juice sample, despite

naturally infected rabbit presented lower IgG levels as compared to

experimentally infected rabbits.

4. Discussion

We described an adequate approach for detection of anti-T. gondii

specific IgG in meat packages, using meat juices with correction for

the amount of blood in different samples and using experimentally

infected rabbits as models for toxoplasmosis. These tests weredescribed elsewhere for detecting antibodies in meat that is usually

diagnosed by inspection for other zoonoses as trichinelosis in meat of

experimentally infected pigs (Kapel et al., 1998; Nckler et al., 2005),

and has been used for detection of Trichinella in pigs from endemic

and non-endemic regions of Europe (Nckler et al., 2004). This

method has been defined as convenient, sensitive and may be used

with confidence (Beck et al., 2005). A good correlation was also

observed for detection of antibodies against Salmonella thyphimurium

Fig. 3. Anti-T. gondii ELISA in meat juice from controls or experimentally infected

rabbits. Open symbols: non-infected rabbits; closed symbols: infected rabbits. Squares

are direct anti T. gondii IgG ELISA absorbance without adjustments. Dots; data

expressed in ELISA (A492nm) determined in a fixed 1:100 blood dilution (cyanhemo-

globin A540nm=0.03). The interruption of y-axis represents the 99% cutoff of the

reaction, based on negative samples.

Table 1

Results of sensitivity, specificity, positive predictive value and negative predictive value

of anti-T. gondii IgG ELISA in meat juice, according to the used assay and experimental

animal origin, as golden standard. C.I. is the confidence interval of 95% of each

proportion according to the numbers of samples tested.

Index Uncorrected ELISA Blood content

Adjusted ELISA

Sensiti vity (C.I. 95%) 72.22% (60.4182.14) 100% (94.40100)

Specificity (C.I. 95%) 100% (85.75100) 100% (92.60100)

Positive predictive value (C.I. 95%) 100% (93.15100) 100% (94.40100)

Negative predictive value (C.I. 95%) 54.55% (38.8569.61) 100% (92.60100)

Fig. 4. Effect of the thawing process and storage in ELISA for anti-T. gondii IgG deter-

mination in meat juices. Meat from the same animal has the same symbol. First thaw is

connected by line to second thaw. Open symbols and solid lines: non-infected rabbits.

Closed symbols and dotted lines: infected rabbits.

Fig. 5. Anti-T. gondii ELISA in meat juice from slaughtered rabbits. The interruption of

the y-axis represents the 99% cutoff of the reaction, using 25 meat juices from cuts of

seronegative rabbits.

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in slaughter pigs (Szab et al., 2008) or anti-Taenia saginata in pig

cysticercosis (Abuseir et al., 2007).

Several studies have described the use of meat juice for detection

of antibodies by ELISA, or by using other methods for toxoplasmosis

diagnosis in the last few years, and this method is considered a useful

and feasible approach in experimentally infected pigs (Hill et al.,

2006; Wingstrand et al., 1997), in retail pork (Dubey et al., 2005),

slaughtered pigs in Sweden (Lundn et al., 2002) and in other market

pigs (Gamble et al., 2005). The promising results were criticized inthese studies due to the absence of any attempts to measure antibody

concentration, resulting in failure, both in sensitivity and specificity

(Dubey et al., 2005).

In our study, we devised a simple method for blood content

determination in meat juice, without additional equipment other than

those necessary for ELISA. We also demonstrate the quantitative

correlation in specific antibodies for anti-T. gondii between serum and

meat juice. Furthermore, immunoblotting revealed similar reactivity

between samples of meat juice and serum, demonstrating that

reactivity of themeat juicesis, in fact, dependent on the concentration

of blood in the tissue and not a local productionor selection of specific

antibodies.

The first and direct approach, as used in most studies (De Lange

et al., 2003; Dubey et al., 2005; Gamble et al., 2005; Hill et al., 2006;

Nielsen et al., 1998; Nckler et al., 2004; Wilhelm et al., 2007) usedthe

direct result of ELISA with a common dilution of meat juice for all of

the samples. As we have shown, this measure may underestimate the

results of positive samples in highly diluted samples, and may

overestimate the results of negative samples, when tested at high

concentrations, due to nonspecific reactions. ELISA had an optimum

range of discrimination. The performance is easily improved if all of

the samples are adjusted to the best ELISA dilution. This solution

results in lowsample numbers with an increased ability to detectanti-

T. gondii IgG in experimentally infected rabbits, or to test rabbit meat

in field conditions, which had lower discrimination than the

experimental models. This preliminary prevalence in rabbit meat

was low as expected in our slaughter rabbits, due to intensive pro-

duction with adequate housing. The lower quantitative data in ELISA

could be explained by natural infection with South American T. gondiitype III strain, which presented lower virulence as compared to type II

strain used in our experimental infection models (Pena et al., 2008).

We are seeking partners for testing commercially marketed rabbit

cuts, which was not attempted in the present study.

Interestingly, this corrected assay was also efficient in meat that

was stored for a prolonged period of time, or in meat subjected to a

second cycle of freezing andthawing,as meat juice hasbeen corrected

to the blood content with similar results. These data were similar to

those described for Aujeszky's disease (De Lange et al., 2003) and for

Salmonella thyphimurium infections in pigs, suggesting that this fluid

can be considered a physiological dilution of serum (Nielsen et al.,

1998). We also demonstrated that the first and second freezing, or

storage time of freezing period of at least 120 days, resulted in similar

results without degradation or interference in the assays. This findingagrees with studies of high levels of antibodies present in meat juice

samples of mice examined using ELISA that were experimentally

infected with Trichinella and then stored for 20 weeks (Wang et al.,

2007).

We reinforce the importance of monitoring programs for toxo-

plasmosis to trace infected animals, as has already been done in many

western countries for Salmonella and Campylobacter sp. (Kijlstra &

Jongert, 2009). Safe meat is mandatory, and the results of such

monitoring will have a direct impact on the income of producers and

on the improvement of management systems. T. gondii tracing is only

casually performed, and it is reported mostly in research projects

(data from the European Food Safety Authority EFSA, 2006).

Monitoring meat by serology could be implemented to identify meat

products asToxoplasma free

. At the same time, meat from positive

animals could be processed and used in pre-cooked products

marketed as Toxoplasma-safe meat.

5. Conclusion

Meat juice is a subproduct of frozenpackagedmeat thatcan easilybe

obtained and can be used in ELISA assay as a source of antibodies to

investigate T. gondii infection in experimentally infected rabbits. This

sample is both non-destructive and obtained from package of meat,allowing adequate quality control. The measurement of the blood

content of meat juices allows the adjustment to a single and stan-

dardized dilution for ELISA, with a better discrimination of positive and

negative samples. Therefore, ELISA can be an alternative to warrant

meat quality or to detect T. gondii infection in imported meats and

from meats that have been previously prepared for marketing, without

tissue destruction or the need to impede further consumption.

Acknowledgements

We would like to gratefully thank the animal colony at Faculdade

de Medicina, USP for providing animals for the experiments, and the

Zootecnia Dept, UNESP, Botucatu, for supplying slaughter samples

from their rabbit zootecnology plant. This work was a part of theMasters in Science program of Juliana N. Mecca, D.V.M., and was

partially supported by FUNDAP, SP, Brazil. This work was also

supported by LIMHCFMUSP-49.

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