Abstract Book of EAVLD2012 - eavld congress 2012

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DETECTION OF EQUINE FOAMY VIRUS INFECTIONS IN HORSES
Magdalena Materniak, Jacek Kuzmak
National Veterinary Research Institute, Department of Biochemistry, Pulawy, Poland
Equine foamy virus, diagnostics, PCR, ELISA
Introduction
Foamy viruses (FVs) are the least known complex retroviruses.
FVs infections are highly prevalent among several animal species
including non-human primates (chimpanzees, gorillas, etc.), cats
and cattle. Another foamy virus was isolated from horses (Equine
foamy virus - EFV) (1). Similarly to other FVs, EFV shows
characteristic ultrastructure and susceptibility to infect large
number of cell lines. Furthermore, its molecular analysis exhibits
genome organization typical for other known foamy viruses, but
its sequence shows the highest similarity to bovine foamy virus.
Although the first isolation of EFV was reported over ten years
ago, still nothing is known about the prevalence of EFV infections
in horses. Therefore we aimed to develop diagnostic tools and
apply them to investigate the EFV infection in horses from
Poland.
References
1. Tobaly-Tapiero J., Bittoun P., Neves M., Guillemin M.-C., Lecellier Ch.-
H., Puvion-Dutilleul F., Gicquel B., Zientara S., Giron M.-L., The H. de,
Saϊb A. (2000). Isolation and characterization of an eqiune foamy virus. J.
Virol., 74, 4064-4073.
2. Romen F., Backes P., Materniak M., Sting R., Vahlenkamp T. W., Riebe
R., Pawlita M., Kuzmak J., Löchelt M. (2007). Serological detection
systems for identification of cows shedding bovine foamy virus via milk.
Virology, 364, 123-131.
Materials & methods
Blood samples were collected from 175 horses (4 -18 years old),
including common breeds, Hucul ponies and randomly selected
samples from slaughtered horses. Semi-nested PCR was
developed and applied to detect EFV DNA in DNA extracted from
peripheral blood leukocytes (PBLs). Selected primers flanked 275
bp fragment within pol gene, the most conservative EFV region.
GST ELISA with BFV specific Gag-GST fusion protein (2) was
used for detection of foamy virus specific antibodies in horse
serum samples. To confirm ELISA results immunoblotting with
antigen prepared from Cf2Th/BFV 100 cells was applied.
Additionally, attempts were made to isolate EFV from peripheral
blood leukocytes of PCR positive animals by co-cultivation with
BHK-21 and RK-13 cells.
Results
12.5% of samples were positive in semi-nested PCR. Specificity
of amplified fragments was confirmed by sequencing and parwise
identity of particular sequences ranged from 95.3% to 98.3%,
when compared to EFV reference sequence (GenBank Acc. No.
NC_002201). In contrast, almost 19% of the samples showed
seroreactivity to BFV Gag protein. Most of PCR positive samples
were detected in Hucul ponies, while only several in samples
randomly collected from slaughtered animals. All attempts to
isolate EFV from PBLs of PCR positive horses failed.
Table 1: Summary of results.
Origin
PCR (+) ELISA (+)
samples samples
Hucul ponies 15 14 0
Saddle-horses 3 6 0
Slaughtered
horses
TOTAL
4
22
13
33
Co-cultivation (+)
samples
Discussion & conclusions
This study presents the first report on prevalence of EFV infection
in horses. The discordance between PCR and serological results
can be due to application of BFV specific tests instead of EFV
specific serological assays. Therefore further studies are
essential to develop EFV specific serological tests to confirm
obtained results. Nevertheless, our study clearly shows that EFV
infections are present among horses in Poland.
Acknowledgements
The authors would like to thank Dr. Ali Saïb and Dr. Joelle
Tobaly-Tapiero for providing EFV positive DNA and serum
controls.
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