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      Detection and dissemination of Toxoplasma gondii in experimentally infected calves, a single test does not tell the whole story

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          Abstract

          Background

          Although the detection of Toxoplasma gondii in bovine tissues is rare, beef might be an important source of human infection. The use of molecular techniques, such as magnetic capture qPCR (MC-qPCR), in combination with the gold standard method for isolating the parasite (mouse bioassay), may increase the sensitivity of T. gondii detection in infected cattle. The risk of transmission of the parasite to humans from undercooked/raw beef is not fully known and further knowledge about the predilection sites of T. gondii within cattle is needed. In the current study, six Holstein Friesian calves ( Bos taurus) were experimentally infected with 10 6  T. gondii oocysts of the M4 strain and, following euthanasia (42 dpi), pooled tissues were tested for presence of the parasite by mouse bioassay and MC-qPCR.

          Results

          Toxoplasma gondii was detected by both MC-qPCR and mouse bioassay from distinct pools (100 g) of tissues comprising: liver, tongue, heart, diaphragm, semitendinosus (hindlimb), longissimus dorsi muscle (sirloin) and psoas major muscle (fillet). When a selection of individual tissues which had been used for mouse bioassay were examined by MC-qPCR, parasite DNA could only be detected from two animals, despite all calves showing seroconversion after infection.

          Conclusions

          It is apparent that one individual test will not provide an answer as to whether a calf harbours T. gondii tissue cysts. Although the calves received a known number of infectious oocysts and highly sensitive methods for the detection of the parasite within bovine tissues were applied (mouse bioassay and MC-qPCR), the results confirm previous studies which report low presence of viable T. gondii in cattle and no clear predilection site within bovine tissues.

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          Most cited references24

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          Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.

          We have identified a novel 529bp fragment that is repeated 200- to 300-fold in the genome of Toxoplasma gondii. This 529bp fragment was utilised for the development of a very sensitive and specific PCR for diagnostic purposes, and a quantitative competitive-PCR for the evaluation of cyst numbers in the brains of chronically infected mice. The 529bp fragment was found in all 60 strains of T. gondii tested, and it discriminates DNA of T. gondii from that of other parasites. Toxoplasma gondii DNA was detected in amniotic fluid of patients, as well as in various tissues from infected mice. Polymerase chain reaction with the 529bp fragment was more sensitive than with the 35-copy B1 gene. For the quantitative competitive-PCR, a 410-bp competitor molecule was co-amplified with similar efficiency as the 529bp fragment. Quantitative competitive-PCR produced a linear relationship between the relative amounts of PCR product and the number of tachyzoites in the range of 10(2)-10(4) tachyzoites and 100-3000 tissue cysts. A highly significant correlation between visual counting of brain cysts and quantitative competitive-PCR was obtained in mice chronically infected with Toxoplasma. Thus, quantitative competitive-PCR with the 529bp fragment can be used as an alternative for the tedious visual counting of brain cysts in experimental animals. With the quantitative competitive-PCR, furthermore, we could confirm the copy number of the 529bp fragment in tachyzoites and estimate the number of bradyzoites per cyst.
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            Prevalence and risk factors for Toxoplasma gondii infection in meat animals and meat products destined for human consumption.

            Toxoplasma gondii is a protozoan parasite that is responsible for approximately 24% of all estimated deaths attributed to foodborne pathogens in the United States. Human infection results from accidental ingestion of oocysts from the environment, in water, or on insufficiently washed produce or from consumption of raw or undercooked meat products that contain T. gondii tissue cysts. This review focused on studies of T. gondii in meat because many human T. gondii infections are acquired through consumption of raw or undercooked meat. Prevalence of T. gondii is higher in conventionally reared pigs, sheep, and poultry than in cattle and is greater in meat products from organic than from conventionally reared meat animals because of outdoor access, which poses substantially greater opportunities for exposure to infected rodents, wildlife, and oocyst-contaminated feed, water, or environmental surfaces. Risk factors related to T. gondii exposure for livestock include farm type, feed source, presence of cats, methods of rodent and bird control, methods of carcass handling, and water quality. This review serves as a useful resource and information repository for informing quantitative risk assessment studies for T. gondii infection in humans through meat consumption.
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              Toxoplasma gondii in animals used for human consumption

              Food-borne toxoplasmosis in humans may result from exposure to different stages of Toxoplasma gondii, in particular from the ingestion of tissue cysts or tachyzoites contained in meat, primary offal (viscera) or meat-derived products of many different animals, or the ingestion of sporulated oocysts that are contained in the environment and may contaminate food and water. Although the potential for transmission of the parasite to humans via food has been known for several decades, it is not known which routes are most important from a public health point of view. It is likely that transmission of the parasite to humans is influenced not only by the potential contamination of various food sources, but also by the individual behaviour of consumers in different ethnic groups and geographical regions. Most current methods for detection of T. gondii in meat-producing animals, in products of animal origin, or in the environment are insufficient because they do not allow quantification of infectious stages. Hence, most studies report only qualitative data from which it is difficult to assess the true risk of infection in individual cases. There is a need for quantitative data so that efficient strategies to reduce food-borne transmission of T. gondii to humans can be developed.
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                Author and article information

                Contributors
                alison.burrells@moredun.ac.uk
                aletaroda@gmail.com
                marieke.opsteegh@rivm.nl
                gereon.schares@fli.de
                cecile.dam@rivm.nl
                paul.bartley@moredun.ac.uk
                francesca.chianini@moredun.ac.uk
                ivillena@chu-reims.fr
                joke.van.der.giessen@rivm.nl
                lee.innes@moredun.ac.uk
                Frank.katzer@moredun.ac.uk
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                18 January 2018
                18 January 2018
                2018
                : 11
                : 45
                Affiliations
                [1 ]ISNI 0000 0001 2186 0964, GRID grid.420013.4, Moredun Research Institute, Pentlands Science Park, ; Bush Loan, Penicuik, Scotland
                [2 ]ISNI 0000 0001 2193 3537, GRID grid.411400.0, Protozoology Laboratory, Departamento de Medicina Veterinária Preventiva, , Universidade Estadual de Londrina – UEL, ; Londrina, PR Brazil
                [3 ]ISNI 0000 0001 2208 0118, GRID grid.31147.30, National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control, ; Bilthoven, The Netherlands
                [4 ]GRID grid.417834.d, Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, , Federal Research Institute for Animal Health, ; - Insel Riems, Greifswald, Germany
                [5 ]Instituto de Ganadería de Montaña (CSIC-ULE), León, Spain
                [6 ]Laboratoire de Parasitologie-Mycologie, Centre National de Référence de la Toxoplasmose, Centre de Ressources Biologiques Toxoplasma, Cédex, Reims, France
                Article
                2632
                10.1186/s13071-018-2632-z
                5774111
                29347971
                1dec6c5b-b7ad-4636-a21a-f9858a34eb63
                © The Author(s). 2018

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 25 October 2017
                : 8 January 2018
                Funding
                Funded by: European Food Safety Authority
                Award ID: GA/EFSA/BIOHAX/2013/01
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Parasitology
                cattle,toxoplasma gondii,food safety,beef,mouse bioassay,magnetic capture,polymerase chain reaction

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