Detection of bovine lymphotropic herpesvirus DNA in tissues of a bovine aborted fetus in Québec Donald Tremblay, Ossama Allam, Richard Drolet and Carl A. Gagnon

Project premises In late 2008, the Molecular Biology Diagnostic Laboratory (MBDL) of the Faculty of veterinary medicine of the University of Montreal started a research project to investigate the etiological agents involved in cow abortion in Québec, Canada -Molecular diagnostic (PCR) 4 principal pathogens: -BVDV -Bovine herpesvirus type 1 (IBR) -Leptospira spp -Neospora caninum 1 more (public health concern): Coxiella burnetti

Data compilation -Analyses are paid for. The results are given to the veterinarian as part of the regular diagnostic (24 hours delay after reception). -The laboratory is sending coolers with identified collecting tubes to the submitting veterinary. -vaccinal status of the herd -live attenuated, -killed viruses and -unvaccinated -Historic -Blood for the serological follow-up

Methodology and results To date (May 2010) -96 cases submitted -86 placentae -PCRs are done on -placentae (when available) -1 pool of all other tissues.

Methodology and results Standard qualitative PCR IBR: polymerase A gene detection Leptospira spp.:Gravekamp C. et al., 1993 Real-Time PCR (quantitative) BVD: AgPath-ID BVDV (Life technologies) Neospora caninum: Ghalmi F. et al., 2008 Coxiella burnetti: Klee S.R. et al., 2006

Results BVDVIBRLeptospira Neospora caninum Coxiella burnetti 5 (5,2)6 (6,3)2 (2,1)9 (9,4)1 (1,0) Number of positive detected by PCR for each pathogen (%)

Results Since 1 case had BVDV and Leptospira spp. -74 samples (77,1%) without any positive detection -abortion rate in the herd? -IBR: Slight increase since January 2010 (4 cases)

Results General bacterial detection by 16S rRNA sequencing: -detection on placenta was inconclusive (environmental contamination of the samples) -Very few amplification on individual tissues (contamination) More target: -Mycobacterium spp. -Salmonella spp. -Toxoplasma -General Herpesvirus -General Coronavirus -Campylobacter spp. -Listeria monocytogenes -Chlamydophila spp. -etc….

General Herpesvirus In January case of BHV4 submitted to the MBDL -First tested for IBR detected with a pan-herpesvirus nested-PCR Vandevanter et al., 1996

General Herpesvirus Exo I Exo II Exo II’Exo III Motif AMotif BMotif C DFAILKTGVIYGKG1 - DNA polymerase gene - Final fragment of 210 bps - Degenerate primers - Product is unique for each viral species (with little strain variation)

Results -First 26 cases were tested for detection of pan-herpesvirus -2 amplifications: -IBR (already reported case) -Bovine lymphotropic herpesvirus (BLHV)

Bovine lymphotropic herpesvirus -Herpesviridae family within the subfamily Gammaherpesvirinae -First described in 1998 (Cornell University) -Rovnak J. et al Search of Herpesvirus as co-factor in the bovine leukemia virus (BLV) pathogenesis - 80% positives in animals tested (n=147)

- Report case in 2006 in the UK - Cobb et al., The Veterinary Report Cases of chronic unresponsive endometritis - 33% of a 180-head milking herd was affected with a purulent metritis that did not respond to standard antibiotic therapies - Investigation in Banks et al., The Veterinary Journal small-scale investigation of UK dairy herds reporting recurrent episodic or chronic non-responsive post-partum metritis -High prevalence in affected herds (but no control unaffected herds) -No association with calving problems Bovine lymphotropic herpesvirus

Results -Second consecutive abortion, fifth in the last year -Placenta and pool of tissues also positive for Neospora caninum -Individual tissues of the aborted fetus tested -Brain and lymph nodes were positive -Tropism of Herpesvirus for neuronal cells and lymphocytes -No histopathological lesion suggestive of a viral infection could be found in BLHV nested-PCR positives tissues

Results Investigation in the 60 other submitted placenta - 6 others BLHV amplified - 1/6 had Leptospira spp. infection -Total of 7 cases (11,7% of 60 submitted placenta)

Conclusion -First description of a Bovine Lymphotropic Herpesvirus in Canada -To our knowledge, it is the first time that BLHV is described in tissues of an aborted fetus -It is difficult to ascertain that BLHV has a role to play in the cause of the abortion (presence of recognized pathogens and no significant viral lesions) -Important to report the presence of BLHV in Canada because it is the first step toward the country improvement of bovine pathogens surveillance.

Questions?