The use of serology to monitor Trichinella infection in wildlife

Transcription

1 The use of serology to monitor Trichinella infection in wildlife Edoardo Pozio Community Reference Laboratory for Parasites Istituto Superiore di Sanità, Rome, Italy

2 The usefulness of serological tests to discriminate between exposed and non-exposed wildlife to Trichinella infection Estimation of the Trichinella prevalence Study of risk factors Evaluation of Trichinella transmission risk between: wildlife and domestic animals wildlife and humans domestic animals and wildlife

3 Request of the Commission Regulation No 2075/ Article 7 Contingency plans The competent authorities of the Member States shall prepare a contingency plan outlining all action to be taken where samples test positive to Trichinella. That plan shall include details covering: investigation of the source of infestation and any spreading among wildlife Article 10 - Inspection of Trichinella-free holdings The competent authority shall ensure that inspections are.recognised as free from Trichinella. The frequency of inspections shall be risk-based, taking account of disease history and. local susceptible wildlife Article 12 - Withdrawal of official recognition of Trichinellafree holdings or regions with negligible risk When information from the monitoring programme or the wildlife monitoring programme shows that a region can no longer be considered a region where the risk of Trichinella

4 Request of the Commission Regulation No 2075/ Annex IV, chapter I, Obligations on food business operators k (ii) an annual surveillance programme exists for wildlife susceptible to Trichinella. The programme shall be risk-based and shall be conducted in an area epidemiologically related to the geographical location of the Trichinellafree farms. The programme shall test the relevant indicator species on the basis of previous findings. The results shall show a prevalence of Trichinella in indicator animals below 0.5 Annex IV, chapter II, Obligations on the competent authorities a risk-based wildlife monitoring programme has been put in place in those areas where wildlife and holdings applying for Trichinella-free status coexist; the monitoring programme optimises parasite detection by applying the most suitable indicator animal and detection technique, by sampling as wide a number of animals and taking as large a meat sample as is feasible; parasites detected in wildlife are identified at species level. by preparing a standardised protocol for a wildlife monitoring programme

5 Request of the Commission Regulation No 2075/ Annex IV, chapter II, Obligations on the competent authorities C. The competent authority may decide to recognise a category of holdings as free from Trichinella where all of the following conditions are met: (d) a risk-based monitoring programme for wildlife has been established in accordance with Chapter II(A)(d) of Annex IV D. In addition to the requirements laid out in Annex IV to Directive 2003/99/EC, the initial report and the subsequent annual reports to the Commission shall contain the following information: (ii) the risk-based wildlife monitoring programmes implemented according to part A(d) above, or equivalent information

6 Advantages of the direct detection method to detect Trichinella sp. infections in wildlife the larva detection is an inconfutable proof of the presence of the parasite easiness to collect the muscle samples special precautions to avoid bacterial contamination are not needed muscle samples can be collected from decaying tissues easy to teach hunters on what we need everybody can collect muscle samples a plastic bag is adequate to transport the sample a knife or a scissor are adequate to collect the muscle samples from the carcass

7 Disadvantages of the direct detection method to detect Trichinella sp. infections in wildlife the weight, size and bad smell of muscle samples can be a problem to forward them to the laboratory the risk to introduce other pathogens in the lab the need of skill technicians to recognise Trichinella larvae from other nematode larvae, which can contaminate the meat samples, mainly in shot animals, in which intestinal nematode larvae can be spread in the internal organs due to the damage caused by the bullet problems related to the digestion of muscle samples which have been previously frozen and thawed

8 Advantages of the indirect detection methods to detect Trichinella sp. infections in wildlife to test a large number of samples in few hours the laboratory does not need a large space in the freezer to preserve the samples technicians performing the test do not need to be skill on the knowledge on Trichinella larva morphology the handling of samples is minimal serum and/or meat juice samples can be used for the detection of multiple infections, not only for anti-trichinella IgG, reducing the sampling costs

9 Muscle juice versus serum The collection of serum samples from dead wild animals can be laborious and need some specific equipments (syringe with the needle or a pipette, e.g. a plastic Pasteur pipette, and a vial) which cannot be always present in the hands of hunters or rangers who collect dead animals; The collection of muscle juice is easier

10 Disadvantages of the indirect detection methods to detect Trichinella sp. infections in wildlife the samples (serum or meat juice) can be collected from fresh carcasses only the need of: pipettes or syringes to collect the samples vials to preserve the samples centrifuge to separate the serum from the clotting only skill technicians can collect samples properly difficulties to collect samples on the field, consequently the need to forward the whole carcass to the laboratory the risk of false positive or negative results the need of species-specific conjugates, which frequently are not available on the market no information on the infection level and Trichinella species

11 The use of serological tests developed for domestic animals, for wildlife As a general role, serological tests are first validated in domestic animals and then used in wildlife, but: the test may not perform identically in wildlife animals due to differences in: host serological response exposure to organisms with similar antigens the lack of a species-specific conjugate

12 Next steps towards the development of an indirect test to detect anti-trichinella IgG in wildlife objective criteria should be established on the condition of samples which can be considered suitable for serology sample storage samples should be collected from target wild species together with a large amount of muscle samples to verify the congruence between direct and-indirect tests the first approach may involve samples from wild boars, which is the original species from which the domestic pig originate

13 What criteria to establish the cut-off? on the basis of the OD value and of the cut-off, serum samples can be: negative positive doubtful for a practical use of serology in the future, the use of the doubtful category can generate confusing and not comparable results I suggests to avoid the use of this category and to consider all doubtful sera as positive: it is better to have false positive than false negative!

14 What criteria to establish the cut-off? Cut-off Frequen ncy negative positive 0 1:10 1:20 1:40 1:80 1:160 1:320 1:640 Non-infected Infected Titre

15 Predictive values of the test The predictive values are the probabilities which provide an answer to: what proportion of the test-positive animals are truly infected? what proportion of the test-negative animals are truly non-infected? Estimation of true prevalence from test results: true prevalence = AP + sp 1/se + sp 1 where AP = apparent prevalence = no. of positives/no. tested sp = specificity se = sensitivity Example: apparent seroprevalence 10% obtained using a test with a se of 97% and a sp of 95%. The true prevalence estimate is 4.8%

16 Serum or meat juice The quality of serum samples collected from hunted animals is generally very bad and they collection is expensive and time consuming The only choice is the collection of meat juices from preferential muscles: preferential muscles should be collected from the carcass, cut in small pieces and frozen in a plastic envelop at -20 C frozen muscles should be thawed, a corner of the plastic envelop can be cut, and the muscle juice can be dropped in a conical vial muscle juice should be tested at a dilution ten times less than that of sera (e.g. 1/10)

17 Preliminary data from CRLP - 1 We have collected and tested 683 muscle juices from wild boars hunted in four Italian provinces during the hunting season 2007/08 Wild boar origin Lombardy region, Northern Italy Digestion positive/tested (%) ELISA positive/tested (%) Brescia province 0/200 91/200 (45.5) Tuscany region, Central Italy Florence province* 0/108 (0) 30/108 (27.8) Grosseto province* 0/256 (0) 34/256 (13.0) Sardinia island Cagliari province* 0/69 (0) 9/69 (13.2) Total 0/ /633 (25.9) *Trichinella sp. infection has been never detected in wild and domestic animals of these provinces

18 Preliminary data from CRLP - 2 The cut off has been established at 18% of the 164 samples with an ELISA Index 18, 100 (60.9%) showed an ELISA Index of 18 28% 33 (20.1%) showed an ELISA Index of > 28 38% 21 (12.8%) showed an ELISA Index of > 38 48% 10 (6.1%) showed an ELISA Index of > 48% Not always there was a correlation between the quality of the muscle juice and the Index Of the 100 samples with a value 18 28%, 30 were tested by Western blot using ES antigens No ELISA-positive meat juice recognized the diagnostic bands, stressing the non-specificity of the ELISA results Of the 10 samples with a value > 48%, all recognized the Trichinella specific bands of the ES antigens by Western blot

19 Suggestions on sample collection 2 meat juice selection of muscles of choice for meat juice collection the muscle juice can be collected from muscle samples cut in small pieces of 2-3 g and frozen for 24 h at -20 C in a plastic bag after thawing at room temperature, the muscle juice can be collected from the bottom of the plastic bag or a corner of the plastic bag can be cut and the muscle juice can be drained in a vial

20 Suggestions on sample collection 3 accompanying data host species* age sex locality of origin* geo-referenced points with GPS or longitude and latitude of the locality of origin* landscape data date of sample collection* type of sample* hours or days between animal death and sample collection* sampling procedure* available volume of serum or muscle juice* (important information is marked by *)

21 Suggested future steps - 1 preparation of a database with information on serum and/or meat juice samples placed at the disposal each sample should include at least the following information: the result of muscle digestion (including the amount of digested muscles and the type of muscle/s) host locality of origin type of sample storage condition data of collection/storage total and/or aliquot available volume donor only samples which fulfilled the above reported criteria, may be included in the database

22 Suggested future steps - 2 as negative controls, we need serum samples from wildlife originating from: Trichinella-free areas or from areas with a very low prevalence positive controls can originate from experimentally infected animals We should subtract the background values of meat juices collected from animals of the same species, originating from areas where Trichinella has been never detected A congruent number of meat juices with a positive ELISA and with different ELISA Indexes should be tested by WB to see the congruence between ELISA Index and specificity