We used retrospective samples from 38 animals submitted to our laboratory for routine diagnosis, including 17 aborted foetuses, four stillbirths or unviable calves and 17 aborting cows from four different cattle herds (totalling 500–700 animals). In total, the 26 samples from 17 cows comprised leukocytes (n = 15), vaginal swabs (n = 9) and placental materials (n = 2). Other samples comprised various tissues from foetuses aborted at seventh to eighth month of gestation and from stillborn or unviable calves (Table 1). To increase the possibility of detecting viruses, each sample from the same animal was tested for BoHV-4 by polymerase chain reaction (PCR) assay along with two other frequently detected pathogens in Turkey, namely, Bovine Herpes Virus 1 (BoHV-1) and BVDV.

Extraction of deoxyribonucleic acid (DNA) for all samples was carried out according to Sambrook, Fritsch and Maniatis (1989). Total RNA was extracted using a high pure viral nucleic acid kit (Roche, Germany) according to the manufacturer’s recommendations. Complementary DNA (cDNA) synthesis was performed using the First Strand cDNA Synthesis Kit (Thermo Scientific, United States) according to the manufacturer’s protocol.

The primer sets specific for gB of BoHV-4 F: (5’-CCCTTCTTTACCACCACCTACA-3’) and R: (5’-TGCCATAGCAGAGAAACAATGA-3’) (Goltz et al. 2004), gC of BoHV-1 F: (5’-CTGCTGTTCGTAGCCCACAACG-3’) and R: (5’-TGTGACTTGGTGCCCATGTCGC-3’) (Van Engelenburg et al. 1993) and 5’-UTR of BVDV F: (5’-ATG CCC WTA GTA GGA CTA GCA-3’) and R: (5’- TCA ACT CCA TGT GCC ATG TAC -3’) (Vilcek et al. 1997) were used in the PCR techniques that are described elsewhere with some minor modifications (Wellenberg et al. 2001).

Specific PCR products (615 base pair (bp), 397 bp and 288 bp of BoHV-4, BoHV-1 and BVDV, respectively) were visualised in transilluminator after electrophoresis in 1% agarose gel containing 10-mg/mL ethidium bromide.

This study was performed within the Scientific Research Projects of Ankara University (Project No.: 10B3338005).

Bovine herpes virus 4 is a co-factor in cattle abortion cases along with other pathogens, including viruses, bacteria and protozoans. They also play a clear role in certain other reproductive disorders such as endometritis (Chastant-Maillard 2015; Cvetojević et al. 2016; Frazier et al. 2002; Reed, Langpap & Bergeland 1979). Researchers have, therefore, focussed on detecting BoHV-4 in semen, uterine and blood samples from aborting cows in herds with postpartum metritis (Cvetojević et al. 2016; Dağalp et al. 2012; Nikolin et al. 2007; Nikolin, Vesna & Radosavljević 2008). However, the connection between BoHV-4 and abortions remains unclear because the virus has been isolated from both diseased and healthy cattle (Czaplicki & Thiry 1998; Deim, Szeredi & Egyed 2007; Leboeuf 2013).

Our results (Tables 1 and 2) show that BoHV-4 infection has a very high prevalence in the samples obtained from Herds I and II. Because very few animals were sampled from Herds III and IV, we could not analyse the prevalence of BoHV-4 infection. However, our previous results and knowledge from veterinarians suggest that BoHV-4 infection is also increasing significantly in these herds, resulting in an increase in economic losses (Bilge-Dagalp 2007, 2008, 2011; Dağalp et al. 2012).

The pathogenesis of BoHV-4 infection has been questioned because of the detection of BoHV-4 from both healthy individuals and cattle with a wide variety of clinical signs (Monge et al. 2006; Wellenberg et al. 2000). In our study, out of 38 animals tested by PCR targeting the gB gene region of BoHV-4, 25 animals, including aborting cows (n = 14) and aborted, stillborn and unviable calves (n = 11), tested positive for the virus. Because of the use of retrospective samples, different materials (blood, vaginal and placental samples) could be tested for aborting cows. However, it is not yet possible to say which of these materials should be used to diagnose BoHV-4 infection, although vaginal specimens appear superior to blood samples (Table 1). Similarly, several tissue samples from an aborted foetus gave positive results for BoHV-4, which indicate that BoHV-4 is transferred to the foetus transplacentally. In addition, isolation of BoHV-4 from a brain sample of a stillborn calf (animal No. 2 in Table 1) in another study (unpublished data) is very important for showing transplacental transfer and the virus’ causative role in fertility problems. In short, our study has substantially determined a link between fertility problems such as abortion and stillbirths and BoHV-4 because we detected the virus in various foetal tissue samples, while the other studied pathogens, especially BVDV, were absent in most of the aborted foetuses (Tables 1 and 2). Verna et al. (2012) likewise concluded that the detection of BoHV-4 as a sole agent offers indirect evidence of the virus’ involvement in bovine abortion.

Several reports indicate that BoHV-4 has an immunosuppressive effect (Egyed 2000; Egyed et al. 1996) and contributes to disease development by stimulating inflammatory reactions (Donofrio et al. 2005). Szenci et al. (2016) reported that local multiplication of Histophilus somni may produce certain chemicals (PGE2) that reactivate latent BoHV-4 from some local macrophages, while multiplication of the reactivated virus decreases the efficiency of local macrophage functions (opsonisation, antigen presentation and killing activity), which further promotes local bacteria multiplication. For this reason, we also tested all our samples for BVDV and BoHV-1, which are very common infections in Turkey, as well as for BoHV-4. The rate of detection of BVDV was as high as for BoHV-4 in Herd I, which had an acceptable sample size of aborting cows and foetuses for this assessment (Table 2). One of the six aborted foetuses was also co-infected with BoHV-4 and BVDV. However, it is not possible to conclude whether this signifies an interaction between BVDV and BoHV-4 infection in these cases, although BoHV-4 has been identified as an important factor affecting the immune system. This could explain the high rates of these infections in Herds I and II. Based on our knowledge, few studies have investigated clinical cases, including abortions and the effects of BoHV-4 along or together with these other viruses. Yilmaz, Coskun and Sahin (2016) had reported that 66.66% of aborted calves (8/12) were positive for BVDV, although BoHV-1 and BoHV-4 were not detected in the sampled animals. Similarly, another study (Tuncer-Göktuna et al. 2016) using PCR analysis for investigating the role of herpes viruses and pesti-viruses in cases of ruminant abortion between 2007 and 2015 in western Turkey did not find BoHV-4 in any of the tested samples from 60 aborted foetuses, while two and 31 calves aborted analysed using ELISA were found positive for BoHV-1 and BVDV antigens, respectively. However, some other studies have reported the presence of BoHV-4 and its co-infection with other pathogens (Cvetojević et al. 2016; Frazier et al. 2002; Reed et al. 1979). Cvetojević et al. (2016) detected BoHV-4 in 21% of examined samples (21/100), co-infection of BoHV-4 with BVDV in two samples and one sample infected with Neospora caninum. Otherwise, several studies have proposed that BoHV-4 is a secondary agent, often associated with secondary bacterial infections (Chastant-Maillard 2015; Donofrio et al. 2008; Klamminger et al. 2017; Jacca et al. 2014; Nak et al. 2011) and also fungi or other viruses (Drolet, Werdin & Goyal 1986; Fabian et al. 2008; Frazier et al. 2002). Gagnon et al. (2017) suggested that because BoHV-4 is a frequent risk or a secondary factor in cattle infections, higher BoHV-4 seroprevalence in cattle with respiratory or reproductive diseases could be expected. Thus, our data detecting two viral pathogens in the same aborted foetus, stillborn calves and aborting cows (Tables 1 and 2) are not surprising. Unfortunately, we were unable to test both foetuses and their mothers. Therefore, more detailed investigations are needed to establish a clear link between BoHV-4 infection and abortion as well as the potential interaction of BoHV-4 with other abortifacient pathogens in the same sample.

In this study, there were two main limiting factors. Firstly, it did not test for the presence of any additional microorganisms (bacteria, etc.). Secondly, it did not include serological results to show the positivity rate for BoHV-4 or the other studied viruses in aborting cows and/or healthy cattle in these herds.

In conclusion, we consider that BoHV-4 alone or in conjunction with other pathogens, especially BVDV, contributed to the development of the reproductive disorders reported in this study, and possibly to others as well. Further studies using epidemiological data from BoHV-4 infections and molecular characterisation of field BoHV-4 in different clinical cases are needed to understand their pathogenesis and responsibility for economic losses affecting cattle husbandry in Turkey.