Diagnostic methods for Toxoplasma gondii: state of the art and future challenges

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1 Thirteenth Workshop of National Reference Laboratories for Parasites Rome, May, 2018 Diagnostic methods for Toxoplasma gondii: state of the art and future challenges Prof. Laura Kramer Department of Veterinary Sciences University of Parma Parma, Italy

2 PRESENTATION OUTLINE Why do we need to diagnose Toxoplasma gondii in food animals How is diagnosis currently carried out In vitro culture as an alternative to bioassay

3 Why do we need to diagnose T. gondii? Prevalence studies Anti-T. gondii antibodies were present in a substantial proportion of breeding pig herds in Estonia.

4 Why do we need to diagnose T. gondii? «Risk» studies: for example, which food animals (and why) are more exposed and thus which products represent a greater consumer risk? T. gondii infection is prevalent in indoor housed Dutch dairy goats but at a lower overall animal level seroprevalence than outdoor farmed goats in other European countries, and cat exposure is an important risk factor.

5 Why do we need to diagnose T. gondii? As a differential diagnostic tool in cases of abortion The importance of veterinary investigation and of reaching a diagnosis is also relevant when considering the different measures that could be taken in the face of an abortion.. (Toxoplasma, Chlamydia, Schmallenberg virus, listeriosisand Q fever)

6 Current methods I. Serology: what s out there for routine use? Da Wyrosdick and Schaefer, 2017, Animal Health Research Reviews

7 Current methods I. Serology: pitfalls and solutions A. Cross reactivity (from Gondin, Mineo and Schares, 2017 Parasitology) Most currently available serologic tests for T. gondii may show some level of cross-reactivity with related coccidia, in particular with H. hammondi and N. caninum. Serological cross-reactivity between T. gondii and N. caninum has been observed when crude antigen ELISAs are employed, but not with recombinant antigens. B. Validation of tests (from Basso et al, 2013 IJP) Difficulty of determining true sensitivities and specificities without a reliable gold standard for comparison (including bioassay) Necessary to perform Bayesian latent class analysis, which does not rely on the assumption of a perfect reference test but rather estimates the accuracy of the candidate test and the reference standard (s) with joint test results. these statistical models are complex and require statistical assistance. C. Best tissues for meat juice serology (Meemken et al, 2014 Prev Vet Med) Heart is a juicy predilection site in pigs and small ruminants (chickens, horses), but are not always easily obtained Validation on tissues, especially diaphrams?

8 EURL Proficiency Testing for T. gondii serology 2015, 2016, 2017 In 2015, 1 laboratory failed, with 3 false positives The cause of laboratory failure has been analyzed and can be attributed to the lack of the method (in house developed agglutination test) set up for ovine serum samples. In 2016, 1 laboratory failed with 3 incorrectly identified samples (pos or neg?) The most frequently used commercial kits were immunoenzimatic assays. All NRLs but laboratory B correctly classifled, as positive or negative, all serum samples. Laboratory B reported problems during last step (reading) of the test (DAT Biomerieux) for two serum samples from the panel. In 2017, no lab failed!!!

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10 Current methods II. Molecular-based approaches direct detection of DNA by PCR establishes the presence of the organism in the tested tissue two most commonly used targets: B1: slightly less sensitive, but does not occur in N. caninum 529 repeat element: more sensitive, most frequently used Limits to molecular identification include: tissue to be sampled The present work showed that the sensitivity of the T. gondii 529 bp RE-PCR assay was slightly higher than the B1-PCR in naturallyinfected swine. However, due to the negative results obtained for a rate of the B1-PCR positive samples, a multi-target PCR approach might appear to be the best approach to improve the reliability of infection in swine, and could also be exploited in food testing. limited amount of target DNA to digest or not to digest.

11 Current methods II. Molecular-based approaches Which tissues? Pigs, small ruminants, horses, poultry: heart, brain Cattle:?? Which technique? Magnetic capture-pcr (from Opsteegh et al, IJFM 2010) combines homogenization of a large sample with sequence-specific magnetic capture extracted DNA is incubated with biotin-labelled capture-oligonucleotides, then added with streptavidin beads, followed by magnetic capture..captured DNA is subjected to PCR LAMP (Loop-mediated isothermal amplification; from Zhuo et al, 2015, VetPar) «LOOP-mediated» refers to the dumbell-like structures that are formed when the multiple primers (inner, outer, loop) amplify the targets able to amplify very small amounts of DNA in a short time «naked-eye» visualization

12 In vitro culture as an alternative to bioassay Why? From the 2016 EFSA report on Experimental studies on Toxoplasma gondii in the main livestock species: With currently available serological methods, implementation of serological screening to identify high risk herds or animals is not considered useful for cattle and horses. For pigs, poultry and small ruminants serological screening can be used to identify high risk herds or animals. However, a negative result in an indirect test can not be used to declare that the meat is safe. PCR positivity confirms the presence of parasite DNA, but says nothing about viability Bioassays are expensive, time consuming and, according to many, unethical (and may even be banned eventually )

13 signed by 1.17 million citizens asks that Directive 2010/63/EU1 on the protection of animals used for scientific purposes is abrogated and that the EU put forward a new proposal aimed at phasing out the practice of animal experimentation The Commission shares the Citizens' Initiative's conviction that animal testing should be phased out. This is the ultimate goal of EU legislation.

14 Is it necessary to confirm viability of T. gondii tissue cysts in meat? From Kilstra and Jongert, 2008 Trends in Parasit

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16 Experience #I: naturally exposed sheep 50 g of tissue from 11 hearts of sheep showing the highest S/P% values on mj serology (ID Vet multi ) Digested, washed, resuspended in 10ml 1.5ml aliquots seeded onto Vero cells, left 3 hours, removed and subjected to extraction and qpcr (529 bp;t0) 5/11 positive at T0 2/11 positive at T1 and T2 No bioassay to confirm results At 7 days (T1) and 21 days (T2), cells removed and subjected to extraction and qpcr

17 Experience #II: pigs and Parma ham X 7/12 8/12 No bioassay on fresh thighs

18 Is in vitro isolation and quantification a valid alternative to bioassay? Pros Cons Ethical Relatively inexpensive Relatively fast (7-21 days) Experienced personnel Ideally, need for positive controls (i.e. experimentally infected animals, i.e. mice?) No application for Toxo-free food Ideas for the future: identification of targes to use as «biosensors of live parasites in digest? Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities.. (Watts et al, 2015 Mbio)

19 Concluding remarks Concordance between direct vs. indirect diagnosis ranges from «none» to «moderate», indicating that currently there is no way to declare food «Toxoplasma-free» The development of new methods that provide an indication of the presence of parasites is considered a research priority, as current direct detection methods are not feasible for large-scale testing. The trend towards phasing out animal experimentation makes the development of alternative models vs. bioassays mandatory.

20 Thanks Edoardo Pozio Anja Joachim Peter Deplazes Walter Basso