A herd of 18 Shetland ponies raised in the Netherlands and transiting through Belgium (Province of Limburg) were found infected by Streptococcus equi subsp. zooepidemicus, a bacterium associated with upper airway diseases in equines and to be differentiated from the

agent of strangles, S. equi subsp. equi, as confirmed by bacteriological analysis of nasal swabs.

 

Interestingly, one of the infected animals had been testing strongly positive for glanders by the Complement Fixation Test and 2 ponies from the same herd tested weakly positive during a routine serological control for export performed on 6 Jul 2011 at the Belgian National Reference Laboratory (NRL). Upon blood resampling performed 14 days later, nasal discharge and enlarged submaxillary lymph nodes were found on the strongly reactive animal. CFT reaction remained positive in the latter animal but had subsided in the weakly reactive ones. The whole herd was put under quarantine by the national authorities.

 

Positive glanders CFT reactions were confirmed for the 3 suspect animals by the OIE reference lab in Jena (Germany) on both sample sets. However, positive serological status could not be confirmed by Western blot in any of the CFT-positive ponies. Western blot is a highly specific serological method used by the OIE reference lab to confirm glanders suspicions in CFT-positive animals. One month after the 2nd control, a 3rd herd sampling was conducted which yielded 4 animals testing positive in the glanders CFT (OIE ref lab). 2 of these showed mild nasal discharge and swollen submaxillary lymph nodes. Bacteriological analysis of the nasal discharge revealed massive contamination by S. equi subsp.  zooepidemicus. Identification was achieved by using both biochemical identification gallery (API 20 STREP, API 20 STREP, BioMrieux) and MALDI-TOF mass spectrometry (Bruker Daltonics BioTyper). Quarantine was released at this point.

 

According to the national authorities, the ponies were born and raised in the Netherlands, had no at risk travel history and had no contact with animals originating from countries where glanders is endemic. They were transiting through Belgium for export outside of the European Union. Bacteriological and molecular analysis (PCR) performed on nasal swabs repeatedly failed to demonstrate the presence of the causative agent of glanders (Burkholderia mallei).

 

False-positive glanders serology has occasionally been reported in animals infected by Streptococcus equi subsp. equi (see for instance Dahmen H., Lehrbuch der Veterinär-Mikrobiologie, 4th edition, Paul Parey Ed., Berlin 1949). However, the molecular basis of the cross-reactivity with members of the Streptococcus equi complex in equids, if any, remains to be elucidated.

 

Inter-laboratory variations in glanders CFT titers originate from the absence of an international standard serum and the use of antigens of different strain blends in individual laboratories.

 

Acknowledgements:

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M. Govaerts and C. De Smedt (glanders reference lab, Brussels)

P. Butaye, M. Van Hessche and H. Van der Veken (general bacteriology,

Brussels)

C. Rettigner, J. Hooyberghs (Federal Agency for the Safety of the Food

Chain, Brussels)

H. Rodriguez-Villalobos and M. Delme (St-Luc Hospital, Brussels)

H. Neubauer and F. Melzer (OIE ref lab, Jena, Germany)

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Pierre Wattiau, PhD.

Veterinary & Agrochemical Research Centre (VAR - CODA - CERVA)

Department of Bacterial Diseases Section of Highly Pahtogenic &

Foodborne Zoonoses (Head)

Groeselenberg, 99 B-1180 Brussels Belgium