An assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death

Numerous viruses, including bovine viral diarrhoea virus (BVDV), bovine herpes virus 1 (BoHV-1) and bovine herpes virus 4 (BoHV-4), and other pathogens are the most common causes of reproductive disorders and are responsible for huge economic losses in livestock production. This study investigates the aetiological role of BoHV-4 in fertility problems such as abortions, stillbirth and birth with unviable calves. Retrospective samples from 38 animals, including 17 aborting cows, 17 aborted foetuses, three stillborn calves and one unviable newborn calf were analysed. The BoHV-4 genome was detected in 25 (65.7%) animals by polymerase chain reaction. In 14 of these infected animals, we detected co-infection with BVDV, while the co-presence of BoHV-1 was also detected in one animal. In addition to the high prevalence of BoHV-4 genome in materials related to fertility problems, isolation of BoHV-4 from the brain of one stillborn calf indicated a causal link between BoHV-4 and fertility problems, such as abortion, stillbirths or birth with unviable calves.

The present study investigates the presence of BoHV-4 genome in samples from aborting cows, aborted foetuses, stillborn calves and unviable calves to investigate the interaction between BoHV-4 and these fertility problems. It also investigated whether there is any significant association between co-infection of BoHV-4 and two other abortifacient pathogens, BVDV and BoHV-1.

Sampled animals
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.

Viral nucleic acid extraction and polymerase chain reaction
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.

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

Results
The PCR results are shown in Table 1, including the description of animals and related materials. Out of 38 animals, the BoHV-4 genome was detected in 25 (65.7%) animals, with 14 (36.8%) and one (2.6%) co-infection rates for BVDV and BoHV-1, respectively. Moreover, out of 38 animals, 19 (50.9%) animals were found to be positive for BVDV and one (2.6%) animal was found to be positive for BoHV-1 ( Table 1). The estimated rate of BoHV-4 positivity was 76.1% (16/21) for aborted foetuses and stillborn or unviable calves and 82.5% (14/17) for cattle (Table 2). In total, 78.9% (30/38) of the sampled animals were positive for at least one of the tested viruses. The data showed that almost all tissues from an aborted foetus with BoHV-4 were found positive (Table 1).
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 2007Dağalp 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 Foetus and others (n = 21) 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 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.