Scientific experts identified a new Actinobacillus pleuropneumoniae (App) serotype 19 and updated the established multiplex PCR (mPCR) for capsule-specific typing (“molecular serotyping”) of all known App serotypes (Stringer et al. 2021a). Primers for species identification based on the apxIV gene were also optimised.

Based on these results it becomes clear that in the recent past some misclassifications regarding the identified App-(capsule)-serotype occurred by means of the cps-mPCR by Bosse et al. (2018), e.g. with App serotype 8 and the so far not identifiable App serotype 19.

Furthermore, the serotype-defining cps locus of App serotypes 9 and 11 is shown to differ only in a single base in the cpsF gene, so that they cannot be differentiated. Since App 9 and 11 also have the same LPS-O antigen locus as well as the same Apx profile, it is proposed that the two serotypes be regarded as hybrid serotypes 9/11.

Retrospectively, we were able to identify a previously untypeable isolate from 2018 as App 19 in our own initial investigations using optimised cps-mPCR (Stringer et al. 2021).

In general, isolates of the same serotype are coding for the same LPS-O-antigens, with some serotypes having the same or very similar O-antigens leading to well-known serologic cross-reactions, e.g. serogroups 3/6/8/15, 4/7 and 1/9/11 (Gottschalk, 2015).

Atypical App variants are characterised by the fact that the isolates differ in biotype (NAD-dependent growth), LPS-O antigen type and/or Apx toxin profile from the respective reference strains of the capsule types (former: serotypes). Recent studies (Schuwerk et al. 2021) show that the proportion of atypical variants may not be insignificant (>70%, but n=4 or 7).

Previous attempts to isolate App in samples and simultaneously characterise the pathogen in terms of serotype and virulence type have largely failed. However, the same renowned research group that newly identified App (serotype) 19 (see above) was recently able to optimise a method in which lung samples from pigs could be typed directly with the new cps-mPCR by Stringer et al. (2021).

For this purpose, suitable lung samples or App-isolates are applied to FTA^® cards (Whatman™). This offers advantages for shipping these samples, as the pathogen is inactivated and therefore no longer infectious when applied to the sample cards. Further, these sample cards do not have to be refrigerated. However, by using FTA^® cards, only DNA is preserved and in limited quantities, whereas archived isolates can usually be accessed again and again through renewed cultivation.

Conclusions for diagnostics:

1. Diagnostics must continue to be adapted to latest scientific findings in the future. It can be expected     that in course of increasing genome analyses further types and variants of App will be identified.
2. According to the current status, all App-isolates should be analysed by the new cps-mPCR by Stringer et al. (2021) to ensure the best possible identification of the capsule type (former: serotype) which is of great importance for the selection of appropriate vaccines as well as for the epidemiologic evaluation.
3. Due to the increasing occurrence of atypic App-variants the virulence potential can be inferred even less than before from the capsule type. It is therefore advisable to additionally establish the Apx-toxin-profile by means of mPCR for all isolates to assess the virulence potential
4. The biotype is determined by classical bacterial cultivation as well as by cps-mPCR using the nadV gene. Confirmation of the App species is ensured by the apxIV target in the cps-mPCR.
5. Capsule-(sero)-typing by cps-mPCR according to Stringer et al. (2021a) directly on suitable lung samples applied on FTA^®-cards has now been validated for the first time (Stringer et al. 2021b).

We will establish and evaluate the procedure and also test it with regard to Apx-toxin typing!

Our diagnostics are state of the art!

The following diagnostic options are available at the IVD GmbH:

• Direct PCR detection of App in suitable sample material using App PCR (App ApxIV PCR) based on the ApxIV toxin gene.
• Culture cultivation and isolation of App isolates from fresh sample material, e.g. after necropsy.
• Molecular serotyping of App isolates by App capsule typing multiplex PCR (App cps-mPCR) updated after Stringer et al. (2021).

• Assessment of the virulence potential of App isolates using App toxin typing multiplex PCR for determination of the Apx toxin profile.

For further information please contact:

Dr. Katrin Stutzberg-Minder

E-Mail: strutzberg@ivd-gmbh.de

Phone: +49 511 220029-0 or -10

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