Introduction
Bovine viral diarrhea (BVD) is recognized as one of the most economically significant diseases of cattle, which affects many cattle-producing countries (Givens et al., 2012). The causative agent is a member of the genus Pestivirus, a group of small, enveloped RNA viruses in the family of Flaviviridae (Yildirim et al., 2011). The disease can lead to embryo resorption, abortion, the birth of weak calves, increased treatment costs, prolonged calving intervals, and reduced milk production (Abbasi et al., 2016).
Various infectious pathogens negatively impact the reproductive system, among which the BVD virus (BVDV) is a significant agent affecting both production and reproduction in dairy cattle. Fetal infection with non-cytopathic BVDV before the development of immune competence can result in early embryonic death, abortion in the last months of pregnancy, or the birth of an immunotolerant calf that is persistently infected (PI). This situation can later necessitate culling to eliminate the virus from the herd (Wathes et al., 2019). 
Epidemiological studies indicate that the virus is widely distributed across various regions and can persist in herds through multiple transmission routes, including direct contact and vertical transmission from dam to calf (Ghaemmaghami et al., 2013; Khodakaram-Tafti & Farjanikish, 2017; Noaman et al., 2020). In a study conducted by Abbasi et al. (2016) 123 cattle (68.33%) were proven seropositive in Sistan and Baluchistan, Iran. Similarly, in another investigation, from 803 collected serum samples belonged to non-vaccinated herds of Arak, 436(54.3%) were seropositive for the virus infection (Ghaemmaghami et al., 2013).
Research has shown that demographic factors, including herd size and density, play a key role in predicting the prevalence of infection in populations where BVD is recurrent (Khodakaram-Tafti & Farjanikish, 2017). Basic principles of infectious disease control rely on two main strategies: eliminating the pathogen’s reservoir and preventing or limiting transmission from infected individuals to susceptible animals, with BVDV being no exception (Walz et al., 2018). Vaccination against BVDV is a common practice in many developed countries (Newcomer et al., 2017). Vaccination can boost immunity in vulnerable cattle populations, helping to prevent clinical disease after exposure to BVDV and reducing the risk of viremia that could result in transplacental infection and the birth of PI offspring (Newcomer et al., 2017). However, vaccination alone does not necessarily lead to complete immunity to infection and abortion. In the study conducted by Wathes et al. in 2019, vaccinated cattle experienced a significant reduction in both abortion and fetal infection rates by nearly 45% and 85%, respectively, but did not achieve complete immunity (Wathes et al., 2019).
The current study aimed to detect BVDV in aborted fetuses from a vaccinated dairy cattle farm using molecular and histopathological methods and to describe other risk factors that should be considered in a susceptible farm despite vaccination. 

Materials and Methods
Study design and sample collection
A total of 23 pregnant cattle lost their fetuses at a gestational age of over four months. Additionally, 2 calves with neurological symptoms were born in the herd. All the aborting cattle and those that gave birth to abnormal calves had received vaccine against BVDV according to the herd vaccination protocol. 
Vaginal swab and abortion discharges were collected from all the aborting cattle. Tissues, including spleen, heart, liver, kidney, lung, small intestine, cerebrum, and cerebellum were taken from 7 aborted fetuses and 2 culled calves with neurological signs. The tissue samples were divided into two parts; one part was collected in sterile urine container for the molecular test and the other part was placed into another urine container with 10% neutral buffered formalin solution for histopathological examinations.

Real-time polymerase chain reaction
RNA was extracted from the swabs and the homogenized tissues using the CinnaGen RNA extraction kit. RNA was reverse transcribed using Superscript II (Invitrogen) and random primers (Amersham Pharmacia Biotech). Real-time polymerase chain reaction (RT-PCR) was conducted according to the recommended method by the World health Organization for Animal Health, 2024. Each reaction included an internal amplification control. Forward and reverse primers and the TaqMan probe used included BVD 190-F 5’-GRA-GTC-GTC-ART-GGT-TCG-AC, V326 5’-TCA-ACT-CCA-TGT-GCC-ATG-TAC and TQ-pesti 5’-FAM-TGC-YAY-GTG-GAC-GAG-GGC-ATG-C-TAMRA-3’, respectively.
Nuclease free water was considered a negative control in all the stages, and the standards of the commercial kit were used as positive controls. Real-time PCR was performed using 2× TaqMan Universal PCR Master Mix, 1 µ concentration of each primer/probe mix, 2 µ tRNA (40 ng/µL), 1.5 µ RNase/DNase-free water, 1 µ 25× enzyme mix, 12.5 µ 2× RT buffer, and sample (or controls). The final volume of the reaction mixture amounted to 25 µ, including 20 µ master mix and 5 µ of sample. 
Amplification was carried out using a Rotor-Gene Q Series (QIAGEN) with the following cycling parameters: heating at 48 °C for 10 min, 95 °C for 10 min (single denaturation step), followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min (annealing and extension). 

Histopathological examinations:
The tissue samples were isolated from each aborted fetus and the slaughtered calves and then, immediately were placed in 10% neutral buffered formalin. The specimens were dehydrated and embedded in paraffin and subsequently, were sectioned with a microtome to obtain 5 µm thick of each tissue. Following deparaffinization and rehydration, the sections were stained with hematoxylin and eosin (H&E) for microscopic analysis. 

Results
The calves with neurological symptoms, including exophthalmos, ataxia, and hydranencephaly (Figure 1) were born in the herd and were found to be BVDV-positive in the molecular method.

None of the fetuses showed gross pathological changes. 

Real-time polymerase chain reaction results
The samples with a cycle threshold (CT) value less than 40 were considered positive. Four out of 7 fetuses (57.14%) and 15 swabs from aborted cattle (65.22%) sent to the laboratory were found positive for BVDV by the molecular test. 

Histopathological results
Pathological lesions attributed to BVDV were visible in the real-time RT-PCR positive samples obtained from the spleen, cerebrum, lung, small intestine, liver, and kidney. Lymphoid tissue depletion was severe in the spleen sections, which was a diagnostic finding for the virus detection. Cerebrum showed multifocal gliosis, but cerebella were normal. Mild vesicular degeneration in the hepatocytes and chronic multifocal purulent interstitial nephritis were visible. The lung sections were indicative of a moderate interstitial pneumonia. Inflammatory response in the small intestine sections, was found by multifocal necrotic cryptitis along with the accumulation of mucus, epithelial debris, and leukocytes. Since BVDV infection leads to the lymphoid tissue depletion along with suppurative and non-suppurative inflammatory responses in the organs, including lung, kidney, intestine, spleen, heart, brain and liver (Montgomery et al., 2008), we strongly suggested that the pathological findings were attributed to BVDV. 

Discussion
The results of the current study indicated a significant abortion rate of 65.22% due to BVD in the cattle vaccinated against the disease, which was confirmed by both molecular and histopathological methods. Histopathological examination which was used on real-time PCR- positive samples to find microscopic changes of BVDV in the fetuses, clearly showed the virus invasion to fetal tissues. Severe lymphoid depletion observed in the spleen, was consistent with the previous studies showing similar findings in peripheral lymphoid tissues due to BVDV-induced immunosuppression (Khodakaram-Tafti & Farjanikish, 2017). Another key histopathological finding noted by Khodakaram-Tafti and Farjanikish, (2017), was multifocal necrotic cryptitis in aborted fetus. Multifocal gliosis in the cerebrum, which was visible in the current study, may represent an early or milder response to BVDV infection, while pseudocyst formation and neuropile degeneration, as described by Montgomery et al. (2008), suggest more advanced lesions. Among all the mentioned histopathological changes, lymphoid depletion and epithelial necrosis of intestine are considered pathognomonic for BVD infections, which were demonstrated in our study.
It is plausible that the real number of positive cases was higher in the current evaluation, as not all the fetuses were accessible for testing due to the factors, such as accidental crushing by heavy machinery of the farm or scavenging by stray dogs. Our findings suggest that vaccination alone may not be sufficient in preventing BVD-induced abortions. Factors other than vaccination affecting the eradication of BVD include biosecurity and control of other diseases (Pinior et al., 2019).
The studied herd exhibited weaknesses in health and management practices despite vaccination. Stray dogs were observed at the farm, and parts of the fetuses’ body, which had been eaten by dogs were found in the cattle’s feed. These factors contributed to the spread of diseases and compromised herd health. Dogs serve as definitive hosts for Neospora caninum, facilitating its transmission and persistence within cattle herds. Studies have demonstrated a potential link between N. caninum infection and the occurrence of BVD (VanLeeuwen et al., 2021). In some cases, infection with the parasite may exacerbate BVD symptoms, including abortions. As reported by VanLeeuwen et al., (2021) co-infections with N. caninum and BVD can increase the severity of clinical signs, such as abortions. In the current study, antibody against N. caninum was detected in 7 of the serum samples and concurrent infection with BVDV was diagnosed in 4 of the aborted animals. As a result, controlling other diseases, especially neosporosis is another factor for eliminating BVD infection. 
The overall hygiene conditions in the studied herd were unsatisfactory, as evidenced by the significant decline in production. Implementing effective hygiene management practices can substantially reduce the risk of BVD outbreaks. Accordingly, good management and biosecurity measures are equally important as vaccination for BVD eradication, and both should be considered components of a control strategy (Moennig & Yarnall, 2021).
Given the high prevalence of BVD in Iran, it is essential to use appropriate vaccine strains and minimize exposure to other pathogens. The significant abortion rate observed in this study shows the detrimental impact of BVD on cattle reproduction. The impact of an outbreak of BVD on reproductive performance and milk production was investigated in 28 Spanish dairy herds using an observational study design, and the group without vaccination showed a higher mean calving-to-conception interval (CCI) and mean number of inseminations per conception (AI/P) (Arnaiz et al., 2021). The reliance of dairy industry on both production and reproduction, lead BVD-induced abortions and neonatal abnormalities caused by the virus to have a substantial negative impact on the economic viability of dairy farms. This is particularly concerning in countries like Iran, where BVD remains endemic (Khezri, 2015). 

Conclusion
To effectively monitor herd health and prevent BVD outbreaks, regular testing of calves is necessary. In addition to employing proper vaccination strategies, implementing comprehensive biosecurity measures is crucial. This includes controlling animal density, using disinfectants, limiting the presence of stray dogs, managing other diseases, and ensuring adequate nutrition. Despite vaccination, a substantial number of cases tested positive, underscoring the critical need for strict adherence to biosecurity protocols along with vaccination.

Ethical Considerations
Compliance with ethical guidelines
This article does not contain any studies with human or animal participants.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Study design: Mona Hamedi; Data collection: Shayan Mofidi and Amir Mohammad Fallah; Writing: Saber Vahedi. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors acknowledge the staff of the studied dairy cattle farm for their assistance in sample collection.


References
Abbasi, J., Hajinezhad, M. R., Sadati, D., Hamshidian, A., Najimi, M., Ghalyanchy Langeroudi, A. (2016). Comparative prevalence of bovine viral diarrhea virus antibodies among native and imported cattle in north of Sistan and Baluchistan- Iran. Iranian Journal of Virology, 10(2-3), 48-52. [Link]
Arnaiz, I., Cerviño, M., Martínez, S., Fouz, R., & Diéguez, F. J. (2021). Bovine viral diarrhea virus (BVDV) infection: Effect on reproductive performance and milk yield in dairy herds. Veterinary Journal (London, England: 1997), 277, 105747. [DOI:10.1016/j.tvjl.2021.105747] [PMID]
Ghaemmaghami, S., Ahmadi, M., Deniko, A., Mokhberosafa, L., & Bakhshesh, M. (2013). Serological study of BVDV and BHV-1 infections in industrial dairy herds of Arak, Iran. Iranian Journal of Veterinary Science and Technology, 5(2), 53-61. [DOI:10.22067/veterinary.v5i2.22723]
Givens, M. D., Marley, M. S., Jones, C. A., Ensley, D. T., Galik, P. K., & Zhang, Y., et al. (2012). Protective effects against abortion and fetal infection following exposure to bovine viral diarrhea virus and bovine herpesvirus 1 during pregnancy in beef heifers that received two doses of a multivalent modified-live virus vaccine prior to breeding. Journal of the American Veterinary Medical Association, 241(4), 484–495. [DOI:10.2460/javma.241.4.484] [PMID] 
Khezri, M. (2015). Bovine viral diarrhea (BVD): A review emphasizing on Iran perspective. Journal of Advanced Veterinary and Animal Research, 2(3), 240. [DOI:10.5455/javar.2015.b92]
Khodakaram-Tafti, A., & Farjanikish, G. H. (2017). Persistent bovine viral diarrhea virus (BVDV) infection in cattle herds. Iranian Journal of Veterinary Research, 18(3), 154–163. [PMID]
Moennig, V., & Yarnall, M. J. (2021). The long journey to bvd eradication. Pathogens (Basel, Switzerland), 10(10), 1292. [DOI:10.3390/pathogens10101292] [PMID]
Montgomery, D. L., Van Olphen, A., Van Campen, H., & Hansen, T. R. (2008). The fetal brain in bovine viral diarrhea virus-infected calves: lesions, distribution, and cellular heterogeneity of viral antigen at 190 days gestation. Veterinary Pathology, 45(3), 288–296. [DOI:10.1354/vp.45-3-288] [PMID]
Noaman, V., & Nabinejad, A. R. (2020). Seroprevalence and risk factors assessment of the three main infectious agents associated with abortion in dairy cattle in Isfahan province, Iran. Tropical Animal Health and Production, 52(4), 2001–2009. [DOI:10.1007/s11250-020-02207-8] [PMID] 
Newcomer, B. W., Chamorro, M. F., & Walz, P. H. (2017). Vaccination of cattle against bovine viral diarrhea virus. Veterinary Microbiology, 206, 78–83. [DOI:10.1016/j.vetmic.2017.04.003] [PMID]
Pinior, B., Garcia, S., Minviel, J. J., & Raboisson, D. (2019). Epidemiological factors and mitigation measures influencing production losses in cattle due to bovine viral diarrhoea virus infection: A meta-analysis. Transboundary and Emerging Diseases, 66(6), 2426–2439. [DOI:10.1111/tbed.13300] [PMID] 
VanLeeuwen, J., Muraya, J., Gitau, G., Makau, D., Crane, B., Mc & Kenna, S., et al. (2021). Seroprevalence and risk factors of Neospora caninum and Bovine Viral Diarrhoea Virus in smallholder dairy cattle in Kenya. East African Journal of Science Technology and Innovation, 3(1). [DOI:10.37425/eajsti.v3i1.391]
Walz, P. H., Riddell, K. P., Newcomer, B. W., Neill, J. D., Falkenberg, S. M., & Cortese, V. S., et al. (2018). Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2. Vaccine, 36(26), 3853–3860. [DOI:10.1016/j.vaccine.2018.04.005] [PMID]
Wathes, D. C., Oguejiofor, C. F., Thomas, C., & Cheng, Z. (2020). Importance of Viral Disease in Dairy Cow Fertility. Engineering (Beijing, China), 6(1), 26–33. [DOI:10.1016/j.eng.2019.07.020] [PMID]
World Organization for Animal Health. (2024). Bovine viral diarrhea. Paris: World Organization for Animal Health. [Link] 
Yildirim, Y., Volkan, Y. Kalaycioglu, A. T., Bilge Dagalp, S., Babaoğlu, A., & Çelebi. Ö., et al. (2011). An investigation of a possible involvement of BVDV, BHV-1 and BHV-4 infections in abortion of dairy cattle in Kars district of Turkey. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 17, 879-883. [DOI:10.9775/kvfd.2011.623]