Toxoplasma gondii antigen for MAT produced in cell culture

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Meat protection and inactivation of T. gondii in meat products

In 1965 prevalence of French population was 83%; 43.8% – 2003; and 37% in 2010 (constantly decreasing). The prevalence always varied substantially according to analysed region in time, population migrations and other complex epidemiological factors (40). A study of Nogareda, Le Strat (101) has predicted the prevalence of T. gondii among French pregnant women to be 27% until 2020. This prediction took into account majority of known risk factors for human infection. Sheep and pigs have traditionally been an important source of meat in many developing countries, and are the only species shown to regularly harbour the parasite (102). This is due to a high resistance of T. gondii in meat and delicatessen. In experimental conditions, bradyzoites in frozen meat survived for 7 days (103), but the temperatures above 58°C killed them after 9.5 minutes (104). When contaminated meat was exposed to pressure bellow 300-400 Mega Pascal (MP), 90% or parasites survived processing (105). In vacuum – packed, not salted goat meat parasites survived for over 6 weeks, while freezing of this meat at – 20°C, inactivated them after 3 hours (106).
One of the most important methods of curing the meat is chemical processing with sodium – chloride, and potassium- or sodium- lactate. Upon penetrating the meat, salts bind free water (water activity – a w), which is essential for parasites survival. Without free water, elementary metabolic processes in parasites can not be performed, exchange of materials with environment is unabled, thus organism’s communication with its surrounding stops. Only 2% of NaCl or 1.4% potassium lactate inactivates parasites within 8 hours (103, 107). Salting in combination with thermic or pressure processing inactivate T. gondii within hours, while salted meat in vacuum protect parasites up to 14 days (106). Inappropriate or insufficient use of these processing techniques allow parasite to survive and stay infectious for animals and humans.

Sampling strategy

In the Netherlands in 2007 different pig farming types were studied independently and the prevalence of 1% on intensive and 5% on outdoor farms was showed (van der Giessen et al. 2007).
Based on the breeding practices reported in the Netherlands and France, the estimation was that T.
gondii prevalence in France could correspond to that of the Netherlands. According to the annual pork production report (Association BD PORC, 2012) the number of samples originating from intensive farms was set at 1,500, with 300 samples coming from outdoor farms. A stratified sampling strategy was devised. Based on the national report of pork production in France for 2012 (Association BD PORC, 2012), eight regions were chosen (Fig 1), representing 92% of the annual pork production in intensive (6 regions) and in outdoor farms (7 regions). In order to obtain the same precision of prevalence estimates on a national level, the same number of samples was collected from each region; from intensive and 43 from outdoor farms, respectively. Next, two levels of stratification were formed: two production types and three age categories. The three age categories were defined as piglets (up to 25kg or 2 months of age), fattening pigs (100-110kg or 8 months of age) and older, breeding pigs-sows. From intensive farms, 115 fattening pigs, 50 piglets and 50 sow samples were collected per region. From outdoor farms, 23 fattening pigs, 10 piglets and 10 sow samples were obtained from each region.  From all eight regions included in the study, 26 slaughterhouses were chosen in relation to the annual number of animals slaughtered. From each abattoir, samples were collected randomly, at least on two different occasions, in order to avoid overrepresentation of samples from the same farm.

Sampling protocol

Heart samples were collected, as described by Villena et al. (2012). All samples were collected from February to December 2013. In agreement with the Ministry of Agriculture (DGAL office – The Directorate for food), all selected slaughterhouses were informed about mandatory participation in this survey. In order to avoid bias and collection errors, the slaughtering dynamics of each abattoir were analysed during the 2012 year. Based on these results, exact dates (periods of intensive production), numbers of samples, and animal categories were determined for sampling in each slaughterhouse.
On the slaughter line, the abattoir veterinarians collected heart samples (minimum 200g) in sterile plastic containers labelled with unique bar codes. Samples were stored at +4°C until the arrival of the transport vehicle, and were then taken (in cooled boxes at +4°C within 12 hours of slaughtering) to the laboratories of the Institute for Animal Health at Maisons-Alfort or the National Reference Centre for toxoplasmosis in Reims. Once in the laboratory, the hearts were stored at +4°C and cardiac fluids were collected for serology analysis (Forbes et al., 2012). Within the following 24 hours, heart digestion was performed on all serology positive animals, and randomly chosen negative ones.

Serology test

Detection of T. gondii antibodies was performed on cardiac fluids by the modified agglutination test (MAT), as described by Villena et al (2012). This is a species-independent serological test, considered to be the gold standard for the detection of T. gondii antibodies in animals and meat (Klun et al., 2006). The antigen was provided by the National Reference Centre for toxoplasmosis in Reims, France. The starting dilution was 1:6, in accordance with a previous study in sheep (Halos et al., 2010). Six further two-fold dilutions were made, up to 1:200. All samples reactive at ≥1:6 were
considered positive.

Mouse bioassay and qPCR

Hearts were digested using trypsin, as described by Dubey et al (1998) and modified by Halos et al. (2010). Briefly, each whole heart was cut and ground slightly. Two-hundred grams were measured and incubated with trypsin at 37°C for 90 minutes (final concentration 0.25%). The suspension was then filtered, centrifuged, and the pellet was washed twice in saline solution. From the total quantity of the pellet, 300 µl were used for DNA extraction and 2 ml were suspended in antibiotics for intra peritoneal inoculation into two Swiss-Webster mice (CD-1) in bioassay (1 ml per mouse. Ethics committee licence no: 21/01/13-2). Mice were bled after 6 weeks from the retro orbital sinus and tested by MAT for T. gondii antibodies. All seropositive mice were euthanized and brains were collected aseptically. Samples of whole brain homogenates were microscopically examined for T. gondii cysts. Of the brain homogenates, 300 µl were used for DNA extraction. All DNA extractions were done using QIAmp mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Quantitative Polymerase Chain Reaction (qPCR), targeting the 529 bp, repetitive element (gene bank accession number AF146527) was performed as previously described by Vujanic et al.,182 (2011).

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Herd selection and risk factors

A written consent from organisations of producers was asked for use of the sera and meta-data in the Toxoplasma gondii seroprevalence survey and risk factors analysis. In Europe, farm prevalence varies from 4% on intensive farms in Netherlands to 85 % in Spain (87, 111). A farm was considered
as T. gondii positive if specific antibodies were found in at least one pig. The expected farm prevalence was set at 20% and relative precision to 5%. The corresponding sample size was calculated to be 60 farms, which were randomly chosen among 125 available from the database. Assuming within-herd prevalence of 5%, the number of samples per farm necessary to detect T. gondii presence at a 95% confidence level was 60 animals. The age of pigs was a single individual risk factor considered, with 4 classes: <1 month, 1-2 months, 2-3 months and >3 months. On all farms pigs are kept in completely confined conditions, without any outside access neither for piglets or sows, separated in buildings identified as farrowing rooms (for sows and piglets from birth to one month of age), weaning (from one to two months old piglets), and fattening roomss (animals from 2-3 months of age are kept in separate pens from older pigs, but there are no distinctive differences in husbandry or hygiene between two age classes). Farm- level risk factors described i) pig housing conditions with three types of bedding (slatted floor, straw, combination of slated and concrete floor), food methods for food distribution (automatic feeders or manual), and ii) hygiene practices, with use of specialized boots and clothes, presence of a Danish entry (yes/no), and the compliance with all-in-all out management (time in days during which pens/rooms/buildings are left empty in post-weaning and fattening processes). All farms used pressurized steam for cleaning and specialized third party always performed rodent control. The number of sows (on a logarithmic scale) was used as a proxy for herd size. Finally the presence of dairy cows or beef cattle, as well as sheep, goats and poultry breeding facilities were considered.

Animal, farm and within-farm levels of T. gondii frequency of seropositivity

All herds had at least one seropositive pig, thus making the frequency of seropositive farms at 100% (95% CI: 94%-100%). When only one age category was observed, farm prevalence in suckling piglets (<1month) was 50% (95% CI: 33%-57%), as well as in weaned animals (1-2 months old – 48%, 95%CI: 27-69%), and 2-3 months old fattening pigs (48%, 95% CI: 35%-62%). However, farm prevalence was higher in pigs older than 3 months: 82% (95% CI: 70%-90%).
The overall animal-level seroprevalence was 7.0% (95% confidence interval [CI]: 6.2%- 7.9%), and varied according to the age class: 5.3% in <1 month old suckling piglets (95% CI: 3.6%- 7.6%), 8.5% in 1-2 months of age weaned animals (95% CI: 5.9%-11.9%), 9.5% in pigs 2-3 months old (95% CI: 7.6%-11.7%), and 6.0% in animals older than 3 months (95% CI: 4.9%-7.1%) (Table 10). Mean within-herd seroprevalence values were very close to the individual-level prevalence, with an overall value of 7.0% (95% confidence interval [CI]: 5.3%-8.6%). This was also true when considering the age classes separately: 5.4% in <1 month animals (95% CI: 3.0%-7.7%), 7.9% in 1 2 months old animals (95% CI: 3.0%-12.8%), 9.5% in 2-3 months old animals (95% CI: 5.0%-14.0%), and 6.0% in animals >3 months of age (95% CI: 4.4%-7.5%).

Table of contents :

Contenu
Remerciements
Résume
Резиме:
Abstract:
Introduction
Biology and epidemiology
Life cycle
Strains and spread-out
Diagnostic
Indirect
Epidemiology in animals
Wild animals
Domestic animals
Pigs
Direct detection of Toxoplasma gondii
Bioassay
PCR based techniques
Strain isolation in Europe
Wild animals
Pigs
Meat protection and inactivation of T. gondii in meat products
Objectives
1. Section 1: Toxoplasma gondii infection in pork produced in France
1.1. SUMMARY
KEY FINDINGS
1.2. INTRODUCTION
1.3. MATERIALS AND METHODS
1.3.1. Sampling strategy
1.3.2. Sampling protocol
1.3.3. Serology test
1.3.4. Mouse bioassay and qPCR
1.3.5. Genotyping of T. gondii isolates
1.3.6. Definition of cases
1.3.7. Statistical analysis
1.4. RESULTS
1.4.1. Collected samples
1.4.2. Seroprevalence analysis
1.4.3. Seroprevalence in pigs from intensive farms
1.4.4. Seroprevalence in pigs from outdoor farms
1.4.5. Live parasite isolation
1.4.6. Concordance between serology and parasite isolation
1.4.7. Risk-factor analysis
1.5. DISCUSSION
2. Section 2: Factors associated with Toxoplasma gondii infection in confined farrow-to-finish pig herds in Western France: an exploratory study in 60 herds
2.1. Summary
2.2. Background
2.3. Materials and methods
2.3.1. Database and serum bank
2.3.2. Herd selection and risk factors
2.3.3. Serology testing
2.3.4. Statistical analysis
2.4. Results
2.4.1. Animal, farm and within-farm levels of T. gondii frequency of seropositivity
2.4.2. Risk factor analysis
2.5. Discussion
2.6. Conclusion
3. Section 3 Working title: Relationship between direct and indirect detection methods of Toxoplasma gondii in naturally infected pigs
3.1. Summary
3.2. Introduction
3.3. Materials and Methods
3.3.1. Pig samples
3.3.2. Indirect detection test – MAT
3.3.3. Direct detection of infectious parasites (mouse bioassay) or parasitic DNA (qPCR and MCPCR)
3.3.4. Statistical analysis
3.4. Results
3.4.1. MAT concordance and correlation between sera and cardiac juice
3.4.2. Direct detection of T. gondii in pigs
3.4.3. Sensitivity and specificity of MAT on sera and cardiac juices
3.4.4. Concordance between direct detection methods
3.5. Discussion.
3.5.1. Cut off analysis for MAT on sera and cardiac juice
3.5.2. Comparisons between MAT on sera and cardiac juices
3.5.3. Concordance results between direct detection methods
3.5.4. Comparison between indirect and direct detection methods
3.6. Conclusions
4. Section 4 Working title: Toxoplasma gondii antigen for MAT produced in cell culture
4.1. Summary
4.2. Introduction:
4.3. Materials and Methods:
4.3.1. Antigen definition:
4.3.2. Mouse antigen preparation:
4.3.3. Cell culture antigen formulation:
4.3.4. Samples for testing
4.3.5. Modified agglutination test
4.3.6. Statistical analysis
4.4. Results:
4.4.1. Antigen production
4.4.2. Concordance:
4.4.3. Correlation:
4.4.4. Cost-benefit:
4.5. Discussion
General discussion and conclusions
Pork production and sampling problems
Breeding type is important for T. gondii prevalence in pigs
Quantitative prediction of pork as an important source for human infection
Prevalence of T. gondii in intensive pig farms from Bretagne: comparison between two studies
Comparison between T. gondii prevalence in pigs, sheep and cattle, three national surveillance
plans in France
Can MAT be useful tool for screening of pigs for T. gondii infection
List of Publications
Scientific articles:
Expert commission participation:
Oral Presentations on international congresses:

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