lmieli@lda22.com; luc.mieli@gmail.com
LDA22 , 7 rue du Sabot, 22440 Ploufragan – France
Abstract
Major keypoints conditioning success of epidemiological studies and eradication plans are discussed on two main levels:
1 Sampling procedures have to
be adapted to PRRS infectiology (biological data),
fit with estimated regional prevalence, and presumed intraherd prevalence (per age),
Preserve sample integrity: particularly, oral fluids must be refrigerated since sampling until analysis in laboratory! (Otherwise we get numerous false negative results)
2 Necessity to fit with reality of analytical performance:
Substantial differences of performance for Elisa and PCR PRRS diagnostic tools on the market points out the need for independent evaluation and approval of Elisa and PCR products (Diagnostic Sp and Se awfully variable)
Introduction
LDA22 is situated in Ploufragan, Northern Britanny, in the heart of main French pig production area, active member of Zoopôle and contributes to independent diagnostics in animal health since many years.
The validation of a test is the evaluation of a process dedicated to determine whether the test is fitting to a particular use or not.
For infectious disease diagnostic tests, the identity and definition of the criteria required for test validation are elusive, and the process leading to a validated test is not fully standardised. Furthermore, for a single pathogen like PRRSV there are numerous and very different needs for using tests in the field, that often require specific validation.
Our purpose is to demonstrate with examples how sampling procedures and lack of independent test validation for PRRSV diagnostics deeply impact the validness in the field for PRRSV control and eradication strategies.
1 How sampling procedures deeply impact and needs to be well elaborated for control and eradication purposes
The sampling procedures include the choice of animal(s), date of sampling, type of samples, number of samples, potential sample contamination, inhibitors (PCR tests, …)
1.1 PRRS infectiology biological data allow to choose among different diagnostic and control strategies.
The overall validity of a strategy even using a validated test is depending on the course of infection (biological data like duration of viraemia, range of sero-conversion (IgG, IgM), etc), and links to clinical/sub-clinical findings :
According to our experience, the period for Ig G seroconversion after PRRSV infection is very often under-evaluated in published works (8 to 12 days post infection), we mainly see this IgG seroconversion between 20 and 30 days post infection in the field. Unpublished data of experimental infection show evidence that seroconversion period depends on infectious dose, ranging up to 20 days.
1.2 Estimated regional prevalence, and presumed intraherd prevalence (per age) are needed to calculate number of herds and samples per herd/per age to be included in the studies.
If using Oral fluids, observe maximum number of 10 pigs per rope! (Otherwise we get numerous false negative results ….)
1.3 Preserve sample integrity: According to our experience, oral fluids must be chilled or frozen since sampling until analysis in laboratory ! (Otherwise we get numerous false negative results ….)
2 How huge diversity of PRRSV diagnostic tests performance (IgG Elisa and PCR) deeply impacts control and eradication programs
2.1 Serological tests
The different parameters that have been investigated are : specificity, detectability, and sensitivity. (reproducibility investigated but not shown, see Mieli and al - IPVS 2002)
2.1.1 Materials and Methods
Four different ELISA kits (A, B, C, and D) were evaluated.
To evaluate specificity, 11 monospecific sera collected from pigs experimentally infected with various viruses and bacteria in AFSSA, Ploufragan, were used:
To evaluate detectability, two different positive sera, one from AFSSA and one from LDA22, Ploufragan, were used as sequential twofold dilutions in negative SPF serum.
Sensitivity was evaluated with the following sera :
10 sera from the field, found positive with the AFSSA reference ELISA technique,
7 sera from the field, from a primary PRRSV infection in a PRRS free area, 14 experimental sera , collected from SPF animals vaccinated with an inactivated vaccine, and found positive by the Merial IFA technique, supplied by Merial, 36 experimental sera, collected from experimental infections with strains of European PRRSV in sows and fattening pigs, supplied by Merial.
2.1.2 Results
•
Specificity:Only one false positive response was observed with ELISA D, in which the monospecific serum directed against PPV was found positive.
•
Detectability:Table 1: Reverse of the highest dilutions that always gave a positive result for positive sera diluted in SPF serum
ELISA kits A B C D
Positive serum from AFSSA 8 32 8 4
Positive serum from LDA22 16 64 4 <4
•
Sensitivity: The ELISA kit D was not tested for sensitivity, due to poor specificityTable 2: Number of sera found positive/number of sera tested
ELISA kits A B C D
Primary infection in the field 7/7 4/7 0/7 0/7
Experimental infection 34/36 26/36 30/36 Not Done Vaccination in SPF pigs
(inactivated vaccine) 2/14 2/14 4/14 Not Done
2.1.3 Discussion
ELISA kit D gave poor specificity results, ELISA kit C did not appear very sensitive. Therefore, the ELISA kits A or B should be preferred to follow PRRSV natural infection in a herd and to establish profiles.
Examples are shown in the oral presentation on practical impact of these evaluation data on grower/finishers serological profiles. These findings established in 2002 still remain valid at this time ….
Field specificity may vary according to the type of animals (boars for example), and confirms experimental results on huge differences between commercial Elisa products.
2.2 PCR tests
Different slides show the same phenomenon for PRRSV PCR tests as for IgG Elisa tests : huge differences between operational performance on Diagnostic sensitivity among different west european strains...
Conclusions
It is not an easy way to elaborate a control or eradication plan for PRRSv :
Concerning laboratory results, we have to deal with sampling difficulties from different origins and the lack of approval for PCR and Elisa commercial diagnostic tools.
References
1. Anonymous (2008) Principles for validation of infectious diseases diagnostic methods in « Manual for diagnostic tests and vaccines – Terrestrial animals », OIE
2. Jacobson (1998) Validation of serological tests for diagnosis of infectious diseases.
Rev. Sci Tech. Off .Int. Epiz. (17) 2, 469-486.
3. Anonymous (2008) Normes de qualité et lignes directrices de l’OIE applicables aux laboratoires vétérinaires : Guide 1 : validation of infectious diseases diagnostic methods - ISBN : 978-92-9044-707-8
4. Epidemiological and diagnostic observations following the elimination of porcine reproductive and respiratory syndrome virus from a breeding herd of pigs by the test and removal protocol. SA Dee and al – Vet Record – 146 – pp 211-213
5. Control an d Eradication of Porcine Reproductive and Respiratory Syndrome. SA Dee and al – Compendium feb 2000
6. Strategies for the control and eradication of PRRS.SA Dee – AVMEC congress Acapulco 2000
7. A comparison of serologic profiles to define PRRS stability in Herds using a commercial vaccine.
A Dean – Proceedings of 15th IPVS congress - Birmingham England 1998
8. Evaluation of different commercial ELISA kits for detecting antibodies against PRRS virus in Europe - L Mieli, N Chenoufi, and C Charreyre IPVS Ames,Iowa, USA 2002 p.363
9. Qualification and evaluation of a new PRRSV IgM IPMA test Mieli L, Baudouard M, Lebon E, Joisel IPVS Hamburg, Germany 2004 Volume 1:127