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In late 2008, the Molecular Biology Diagnostic Laboratory (MBDL) of the Faculty of Veterinary Medicine, University of Montreal initiated a research project to investigate the etiological agents involved in cow abortion in Quebec, Canada. Several reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR-based assays were done on placenta and tissues of aborted fetuses that were submitted to the MBDL for the detection of bovine pathogens that are known to be involved in abortion such as bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheatis herpesvirus (IBR), Neospora caninum, Leptospira spp. and Coxiella burnetii (1,2). From the 26 submitted cases of bovine abortion that were tested, none were positive for BVDV and Leptospira spp., whereas 3.8%, 11.5%, and 3.8% were positive for IBR, Neospora caninum, and Coxiella burnetii, respectively (Table 1).
In addition to the usual PCR assays, a pan-herpesvirus nested-PCR assay, which is able to detect a broad spectrum of herpesvirus species, was conducted on placenta and pooled tissues of aborted fetuses as previously described (3). Briefly, DNA was extracted from samples with the QIAamp DNA mini kit (QIAGEN, Mississauga, Ontario) according to the manufacturer’s tissue protocol. The PCR was carried out in a Biometra T3 thermocycler with the QIAGEN Fast Cycling PCR kit according to the manufacturer’s specification with a set of 3 primers (DFA, ILK, and KG1). Amplification conditions were: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 96°C for 5 s, annealing at 46°C for 12 s, and extension at 68°C for 45 s. Reaction was ended with a final extension step of 1 min at 72°C. Nested-PCR was then performed under the same conditions using 5 μL of the first reaction and 2 other primers (TGV, IYG).
Excluding the IBR-positive cases, one submitted sample (FMV09-1125585) proved positive in the pan-herpesvirus nested-PCR assay (Table 1). Interestingly, PCR fragment sequencing and GenBank comparison by BLAST showed high homology with the DPOL viral gene of a bovine lymphotropic herpesvirus (BLHV), which is a virus classified in the Herpesviridae family within the subfamily Gammaherpesvirinae (4). Since the first PCR assay amplifies a rather small DNA fragment [< 300 base pairs (bp)], and to further characterize the viral genome, the primers DFA and IYG, encompassing a 483 nucleotides fragment of the DPOL gene, were used in a PCR assay. The resulting amplified DNA was sequenced and compared with other herpesvirus DNA sequences using the BioEdit software (BioEdit Sequence Alignment Editor version 18.104.22.168, Ibis Therapeutics; Carlsbad, California, USA) and the CLUSTAL W alignment method. The FMV09-1125585 BLHV DNA sequence (GenBank accession number HM152484) possesses a 100% nucleotides identity with another GenBank reported BLHV sequence (GenBank accession number AF327830) (data not shown). To our knowledge, this is the first time that BLHV has been found in the bovine species in Canada. This virus has been previously reported only in 2 countries; the United States (US) in 1998 (4) and the United Kingdom (UK) in 2006 (5). The prevalence of BLHV seems very high, the virus was identified by PCR in 91% of tested adult animals (n = 101) originating from Colorado, New York, and New Jersey in the US (4) and in 27% of the UK tested samples (n = 66 tested samples) from cows experiencing non-responsive post-partum metritis (NPPM) (6). Interestingly, the prevalence of BLHV in young animals also seems to be very high, the virus having been identified by PCR in 38% of the tested 2-week-old calves (n = 13) (6). Unfortunately, the BLHV transmission mode is unknown.
Since the first assay on a pool of tissues of the aborted fetus gave a negative result and to further investigate the case, individual tissues were tested by nested-PCR and amplicons that were produced were sequenced. Interestingly, brain and lymph nodes were BLHV positive and, to our knowledge, this is the first time that BLHV was detected in tissues of a bovine aborted fetus. Noteworthy, is that BLHV has been previously found by PCR within lymphoma cells, peripheral blood mononuclear cells (PBMC), vaginal exudates, and vaginal swabs (4–6). It is not surprising to have found BLHV DNA in brain and lymph nodes tissues in an aborted fetus since herpesviruses are well known to possess a tropism for neuronal cells and lymphocytes (7). Samples of spleen, liver, kidney, and heart were negative.
With regard to the FMV09-1125585 clinical case, the submitted tissues were from a 2nd consecutive abortion in the herd of 100 animals and the 5th one during a 12-month period. Placenta and pool of tissues were also PCR-positive for Neospora caninum (one of the 3 cases presented in Table 1), which is an important pathogen involved in cow abortion (2). Histopathological findings revealed a mild and multifocal neutrophilic placentitis. In the liver of the aborted fetus, there was marked extramedullary hematopoiesis. Small intrasinusoidal thrombi were found multifocally within the hepatic parenchyma and were often associated with necrosis of a variable number of hepatocytes. There was no significant lesion in the sections of brain, heart, lung, spleen, and kidney examined. Unfortunately, lymph nodes were not submitted for microscopic evaluation.
Even though others have reported the presence of BLHV with bovine leukemia virus (BLV) within tumor cells and in samples originating from dairy cows experiencing NPPM, the involvement of BLHV in the induction of any diseases is still uncertain (4–6). In addition, the BLV and BLHV status of the dam is unknown and no histopathological lesion suggestive of a viral infection could be found in BLHV nested-PCR positive tissues. Thus, it is very difficult to ascertain that BLHV has a role to play in the cause of the abortion. Moreover, the presence of Neospora caninum was confirmed by PCR in the BLHV positive case. Nonetheless, it is important to report the presence of BLHV in Canada because this is the first step toward the country’s improvement of bovine pathogen surveillance.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant (to CAG). The authors are grateful to Dr. Paul Baillargeon (Pfizer Canada) for the submission of bovine aborted fetuses tissue samples to the Diagnostic Service of the Faculté de médecine vétérinaire, Université de Montréal and for covering the cost of the BVDV and IBR PCR assays.
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