Effect of Domestic Freezing on the Viability of Toxoplasma gondii in Raw and Dry-Cured Ham from Experimentally Infected Pigs.

ABSTRACT: Toxoplasma gondii is the causative agent of the parasitic disease toxoplasmosis, which is an important foodborne zoonosis. Eating undercooked meat of infected animals has been considered the major transmission route of T. gondii to humans. The present study was conducted to evaluate the efficacy of domestic freezing for inactivation of T. gondii bradyzoites in ham. Raw and dry-cured ham from a pig experimentally inoculated orally with 4,000 oocysts of T. gondii VEG strain was subjected to domestic freezing at -20°C for up to 14 days. The effect was evaluated by bioassay in mice and a quantitative PCR assay. In raw and dry-cured ham, -20°C for 7 and 14 days, respectively, did not inactivate T. gondii. More studies are needed to find the correct temperature and time needed to render the bradyzoites noninfectious for humans. Meanwhile, the recommendations for freezing to inactivate T. gondii in raw or dry-cured meats must be revisited because domestic freezing conditions do not reduce the risk of infection.

Toxoplasma gondii in raw and dry-cured ham: The influence of the curing process.

The aim of this work was to analyze Toxoplasma gondii in raw hams by mouse bioassay and to evaluate the effect of curing on the viability of the parasite to assess the risk of infection from eating dry-cured ham. After a serology study of 1200 pigs in Aragón (Spain), forty-one naturally infected pigs with different serological titers against T. gondii were selected. Two cured periods (9 and 12 months) were evaluated as well as the influence of the physicochemical composition of hams on T. gondii survival. Although the parasite burden was low, a high number of seropositive pigs with Toxoplasma tissues cysts in raw hams were found (31.6%). Viability of T. gondii was influenced by the curing, with statistically significant differences between fresh and cured hams (p < 0.001). The viability was higher in hams cured for 9 months compared to those cured for 12 months. However, this period of curing resulted in the reduction but not in a complete elimination of the risk. Thus, from a public health point of view, under the conditions of this study it is safer to consume dry-cured ham with periods of curing higher than 12 months. Analysis of physicochemical results did not identify any variable with significant influence on the presence and viability of T. gondii in cured ham, but loss of viability of T. gondii was observed in hams with a lower fat content. Further research is required to validate combinations of salts concentration and time of curing that can be used as preventive measures in the HACCP system of dry-cured ham industry.

Toxoplasma gondii: Pig seroprevalence, associated risk factors and viability in fresh pork meat.

This study was conducted on 161 fattening pig farms located in Aragón (Northeast Spain). Serum samples from 1200 pigs were tested for antibodies against T. gondii by indirect immunofluorescence assay (IFA). Antibodies to T. gondii (≥1:20) were detected in 301 pigs (24.52%). The seroprevalence observed in the present study indicates a widespread exposure to T. gondii, as seropositive pigs were found in 96.67% of the farms studied although low pig titers were determined. Risk factors associated with T. gondii seroprevalence were presence of cats in or around the farms, presence of dogs around the facilities, low number of animals in the farms, poor hygiene and bad maintenance of the farms. Finally, it was observed that where rodent baits were used, Toxoplasma prevalence was lower. Risk management measures including control of cats and rodents on the farms, among others, could help to reduce the observed prevalence levels. By mouse bioassay, T. gondii was detected in 73.7% and isolated from 42.1% of seropositive pigs and a significant relation between the titers of pigs and the presence and viability of T. gondii in the tissues was found. The detection of T. gondii is not possible by currently practiced meat inspection. Nevertheless, the increased probability of detecting viable forms of T. gondii in tissues of pigs with titers ≥1: 80 could be used as the cutoff for discriminating higher risk animals, and could be used as an effective control tool for the industry of cured meat products. In practical terms, we propose that this value could be used as a critical limit in the HACCP system.

Evaluation of heavy metals and polycyclic aromatic hydrocarbons in honeys from different origins.

A survey of honey samples from different geographical and botanical origins, including some samples collected from a fire-affected area in Spain, was conducted to assess their content of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The levels of the determined toxic elements (Pb, Cd, As, and Sn) were low and were in the range of those reported by other studies. In our work the total amount of heavy metals and Pb was higher in dark honeys than in pale honeys. In the collected samples, no detectable levels of the 15 PAHs studied were found. The obtained data served to assess the levels of heavy metals and PAHs in honey samples from different geographical and environmental origins and to contribute to the scarce data about pollutant content of this matrix. In light of these results, the analyzed samples do not pose any serious concern to human health, and the data obtained in this study could serve to contribute to the establishment of specific maximum limits for honey.

Toxoplasma gondii in commercially available pork meat and cured ham: a contribution to risk assessment for consumers.

Toxoplasmosis is an infection caused by Toxoplasma gondii, whose transmission has usually been attributed to ingestion of undercooked or raw meat. Dry-cured ham is a high-quality meat product of increasing economic relevance, and epidemiological studies point to cured meat products as a risk factor for acquiring toxoplasmosis. With the aim of contributing to the risk assessment process, 50 samples of fresh pork meat and commercial cured ham were collected in the city of Zaragoza (northeastern Spain), and the presence of viable forms of T. gondii was analyzed. A mouse concentration bioassay technique was used, and the presence of the parasite in mice was determined by indirect immunofluorescence assay. T. gondii was detected in two samples of rib, reflecting a frequency of 8% positive fresh pork meat (4% positivity of total samples analyzed). Brains of seropositive mice were analyzed by histology and PCR, although the parasite was not isolated in the seroconverted mice. No viable forms were detected either in other types of fresh meat or in the samples of cured ham.

Multiresidue determination of carcinogenic polycyclic aromatic hydrocarbons in honey by solid-phase extraction and high-performance liquid chromatography.

An analytical procedure based on solid-phase extraction, using ethyl acetate as the elution solvent, and high-performance liquid chromatography with fluorescence and diode array detection was developed for the identification and quantification of polycyclic aromatic hydrocarbons (PAHs) in honey. The method has been optimized and validated in accordance with Commission Regulation 333/2007 and Commission Decision 2002/657/EC. This method allows the identification of the 15 PAHs that should be monitored in food matrices, as proposed in 2002 by the Scientific Committee on Food and later by the European Union in the Commission Recommendation 2005/108/EC, because of their genotoxic and carcinogenic properties. The results of the validation study were in agreement with quality criteria described in European legislation in terms of sensitivity, accuracy, and ruggedness, and the method was applied to the analysis of 42 honey samples (21 from Spain and 21 from other regions). The honey samples were not contaminated by PAHs at detectable levels and thus could be marketed without health risk.

Impact of the use of fluvalinate on different types of beeswax from Spanish hives.

Acaricides are applied in agriculture as phytosanitary products against pests and in apiculture to control the bee parasite Varroa destructor. Poor apicultural practices could result in an accumulation of residues in honeybees, in the environment, and in beeswax and other bee products by migration from the wax comb into stored honey through a process of diffusion and consequently constitute a potential risk for humans. In this study, six different types of beeswax samples were analysed for the determination of residues of fluvalinate, coumaphos, and bromopropylate and its metabolite 4,4'-dibromobenzophenone, all of which are the most commonly acaricides used by Spanish beekeepers against V. destructor. The analytic method consists of solid-phase extraction on a SPE Florisil cartridge and high-performance liquid chromatography separation using a photo diode array detector. The results show that fluvalinate residues were detected in 36.3% of samples, ranging from 1.2 to 6.6 microg/g wax. Residues of coumaphos, bromopropylate, and 4,4'-dibromobenzophenone were not found to be greater than their detection limits. This study indicates that the analysis of these compounds in beeswax samples could be used as bioindicators of fluvalinate sanitary treatment and handling practices applied by beekeepers.

Determination of synthetic acaricides residues in beeswax by high-performance liquid chromatography with photodiode array detector.

A multiresidue HPLC method for identification and quantification of the synthetic acaricides fluvalinate, coumaphos, bromopropylate and its metabolite 4,4'-dibromobenzophenone in beeswax has been developed. Different techniques were tested and modified. The method consists of a sample preparation with isooctane followed by solid phase extraction using Florisil columns. Determination of the synthetic acaricides is achieved by HPLC with a photodiode array detector. Analytical performance of the proposed method, including sensitivity, accuracy and precision was satisfactory. The LOD for the analytes varied between 0.1 and 0.2 microg g(-1) wax and the recoveries between 70 and 110%. Relative standard deviation of the repeatability of the method is <15% and reproducibility is <31%.

Evaluation of residues of essential oil components in honey after different anti-varroa treatments.

Apiary trials on the use of three different treatments (Apilife Var, thymol solution in olive oil, and thymol solution in ethanol) for the control of Varroa destructor were conducted in Aragon (northeastern Spain). For the evaluation of the presence of residues of these treatments in honey an analytical method was developed. The method is applied to analyze honey samples before and after treatments with the acaricides mentioned. A solid-phase extraction on trifunctional silane SPE C18 cartridge and gas chromatography separation using a flame ionization detector allow reliable and precise determination of residues of thymol, menthol, eucalyptol, and camphor in honey. The results indicate that camphor is present in only low concentrations, residues of eucalyptol or menthol were not found at all, and only thymol left residues in high concentrations. Residues of thymol found in honey collected from the beehives ranged from 0.75 to 8.20 microg/g for Apilife Var, from 0.03 to 6.30 microg/g for thymol solution in olive oil, and from 0.05 to 6.20 microg/g for thymol solution in ethanol. Even so, natural treatments can be considered to be good alternatives for synthetic acaricides, especially because they do not represent a sanitary risk.

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS.

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.