SOME STUDIES ON BOVINE VIRAL DIARRHEA IN ASSIUT GOVERNORATE, EGYPT

Bovine viral diarrhea (BVD) is a common viral disease that can affect both domesticated and wild animals. Despite their economic losses, there isn't much information available on BVD in Assiut Governorate. Therefore, the present study's objectives were to determine the clinical findings, risk factors associations with the infection rate, and molecular diagnosis of BVD virus (BVDV). The current study was conducted on 50 cattle that belonged to different villages in Assiut Governorate, Egypt. The clinical examination findings revealed fever, oral lesions, diarrhea, respiratory symptoms, and corneal opacity. Serum samples were collected for laboratory analysis. Reverse transcriptase polymerase chain reaction (RT-PCR) assay had been employed for BVDV diagnosis. BVDV RNA was found in the serum of seven cattle. There was no significant difference (P<0.05) between the percentages of BVD infection and the sex, age, and breed (native and mixed breed) of molecularly tested cattle. According to the climatologic circumstances of Assiut governorate, there was a discernible variation (P<0.05) between the BVD infection rate and the cold and hot months. It is advisable to emphasize how crucial it is to implement efficient preventative and control measures throughout Egypt in order to reduce the prevalence of BVDV.


INTRODUCTION
BVD is a widely spread, ubiquitous viral disease that affects domestic animals like cattle, goats, sheep, and pigs, as well as numerous wild and captive animals (Gong et al., 2013;Sharawi et al., 2016).BVD is one of the most serious cattle diseases in the world and has become a global pandemic due to its widespread nature, transmission, and lack of treatment, in addition to causing significant economic losses to the livestock industry (Sharawi et al., 2016 andPaixão et al., 2018).BVDV is a virus of the genus Pestivirus and belongs to the family Flaviviridae (Weinstock et al., 2001).Pestiviruses are enveloped viruses with a single-stranded, 11.3-13.1 kb positive-sense RNA (Porto et al., 2021).Pestiviruses have been given new names, and the International Committee on Taxonomy of Viruses (ICTV) has classified them into 11 species (Afify et al., 2022).Accordingly, they are divided into the following categories: BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), border disease (Pestivirus D), classical swine fever (Pestivirus C), and HoBi-like (Pestivirus H) (Zambotti et al., 2020 andAfify et al., 2022).The BVDV genome possesses a long open reading frame (ORF) bordered at the 5' and 3' termini with a highly conserved untranslated region (UTR) for translation and replication regulations, respectively (Abd El-Hafeiz et al., 2022).In the BVDV genome, the ORF encodes polyproteins that are split into viral and cellular proteases both during and after translation into four structural (C, Erns, E1, and E2) and eight nonstructural (Npro, p7, NS2-3, NS4A, NS4B, and NS5A) proteins (Abd El-Hafeiz et al., 2022 andAfify et al., 2022).The BVDV can be classified into cytopathic (Cp) and non-cytopathic (NCp) biotypes based on the characteristics of their in vitro cell culture and genetic variation (Kučer et al., 2022).The pathogenicity and clinical symptoms of BVDV infection vary as well, depending on the strain (Weinstock et al., 2001).Cattle infected with BVDV may have gastrointestinal, and respiratory problems, immunosuppression with secondary infections, thrombocytopenia, and reproductive failure (Porto et al., 2021).In pregnant cows, transplacental infection with BVDV occurs with a high degree of efficiency and can result in fetal death, malformations, abortion, or the birth of a persistently infected (PI) calf in addition to the acute and chronic mucosal disease (Weinstock et al., 2001 andKučer et al., 2022).The economic repercussions of BVD have led numerous endemic countries, notably Egypt, to start BVDV control or eradication programs (Afify et al., 2022).Detecting PI animals, eliminating them from herds, and limiting the introduction of new PI animals through biosecurity initiatives and/or vaccination are the two main goals of the majority of control programs in many countries (Afify et al., 2022).In Egypt, BVDV was discovered for the first time in a calf with acute enteritis in 1972 (Hafez, 1972).The majority of BVDV reports from Egypt are mostly based on virus isolation and/or viral antibody identification (Soltan et al., 2015).The detection of BVDV by RT-PCR has been proven to be more rapid and sensitive than virus isolation, with 100% specificity and sensitivity (Atwa et al., 2019).Furthermore, unlike viral isolation, the presence of antibodies in serum samples has no impact on RT-PCR results (Atwa et al., 2019).The identification of risk factors associated with BVDV infection became important as determined indicators were utilized to lead the development of preventive and control measures employed to directly lower the spreading of infection and minimize economic loss (Marques et al., 2016).In Egypt, BVDV is still a concern in the cattle population in several governorates despite control measures like vaccination (El-Bagoury et al., 2014).There is little information about BVD in Assiut Governorate, so the present study aimed to molecularly detect a highly conserved portion of the 5' UTR of the BVDV genome based on RT-PCR, evaluate the clinical manifestations, and study the correlation between some risk factors, such as sex, age, breed of cattle, and seasonal variation, with the infection rate of BVD in Assiut Governorate.

Animals and Ethical approval
Throughout the investigation period, from March 2022 to March 2023, a total of 50 cattle of various sexes, ages, and breeds that came from various villages in Assiut Governorate were admitted to the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Assiut University.All the tested cattle were suffering from clinical signs suggesting their infection with BVDV.All cattle used in this study were handled in accordance with ethical considerations.The study was approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt; the approval number was 06/2023/0085.

Clinical examination
The Clinical examination of the investigated diseased cattle was carried out in accordance with (Jackson and Cockcroft, 2002).

Sampling
Four milliliters of blood samples were collected from 50 clinically diseased cattle that suffered from fever, oral lesions (ulcers), diarrhea, respiratory symptoms like nasal discharge and cough, and corneal opacity into sterile plain vacutainer tubes without anticoagulant through the jugular vein while the animals were adequately held.Blood samples were allowed to clot and centrifuged at 3000 r/min for 20 minutes, and serum was delicately collected (Afify et al., 2022) and stored in separate tubes at -20°C for subsequent RNA extraction.

Molecular diagnosis 4.1. Viral RNA extraction
The viral RNA was extracted from 50 serum samples using the EasyPure® Simple Viral DNA/RNA extraction kit (TransGen Biotech, China), according to the instructions of the manufacturer.

Primers
The specificities of the chosen primers (Metabion International AG, Germany) used in the current study for the 5'UTR of BVDV had been evaluated previously (Weinstock et al., 2001).Sequences of primers and their locations in the viral genome are displayed in Table 1.

Statistical analysis
The statistical package for the social sciences (SPSS) version 16 software was used to enroll and analyze the collected epidemiology data using the Chi-square of independence (2007).

Field diagnosis of clinical cases infected with BVD
Cattle with BVD were diagnosed in the field based on clinical examination and the identification of characteristic clinical symptoms.Cattle used in this study had the typical clinical signs of BVD, such as fever, oral lesions, diarrhea, respiratory symptoms, and corneal opacity.The presence of oral lesions, like ulcers, was the most distinctive feature in 6 clinical cases of infected cattle with BVD (Table 2 and Fig. 1A).Fever (40-40.2o C) was found in 5 of the tested cattle (Table 2).Diarrhea and respiratory signs like a cough and nasal discharge were detected in 3 and 2 of the investigated cattle (Table 2 and Fig. 1B), respectively.One cattle suffered from bilateral corneal opacity (Table 2).

Relationship between the percentage of infection of BVD and potential risk factors 3.1. Percentage of BVD infection
The current study found that 14% (7/50) of the cattle under investigation were infected with BVD (Table 3).

Effect of sex
The analytical findings showed that there was no significant variation in BVD infection percentage between the male and female examined cattle, although a greater infection rate in males than females numerically (Table 3).

Age susceptibility
The prevalence of BVD infection was detected in cattle at the ages of 3 months-1 year, >1-3 years, and >3-5 years, with results showing that, out of 50 animals examined, 20%, 7.41%, and 25% had the infection.Age groups >3-5 years had the mathematically greatest rate of BVD infection (Table 3).

Breed susceptibility
The percentage of BVD infection in native and mixed breed cattle in the current investigation did not significantly differ, despite the higher infection rate in native breeds being more accurate numerically (Table 3).

Seasonal variation
The percentage of BVD infection was significantly higher in hot months (26.32%) than in cold months (6.45%), according to our findings (Table 3).(2020).The confirmation identification was carried out using the RT-PCR method, which is still widely recognized as the best, most sensitive, specific molecular assay for detecting BVDV in clinical samples and allowing for the detection of animals that are transiently infected when the virus load is low (Weinstock et al., 2001;El-Bagoury et al., 2014;Soltan et al., 2015 andChang et al., 2021).The primers used in this study targeted the 5'UTR region, were highly conserved, and were capable of detecting a variety of bovine Pestiviruses, including BVDV-1, BVDV-2, and BVDV-3 (Kosinova et al., 2007 andChang et al., 2021) Some of the risk factors including sex, age, breed of cattle and seasonal variation were investigated for their interaction with the BVD infection rate in the current study.Despite the actual means by which these factors biologically interact with BVD infection requires further investigation, the data obtained suggests that not all these factors may play a significant impact on the BVD infection rate.Concerning sex susceptibility, there was no statistically significant variation in the rate of BVD infection between the male and female investigated cattle, despite the higher infection rate in males than females numerically.Our result concurred with Wilson et al. (2016), who reported that there were no significant differences in BVD infection by sex of cattle.This finding contrasted with that of Daves et al. (2016), who reported that females had a higher prevalence of BVD than male cattle.Our result could imply that BVD is a non-sexrelated disease and that male and female cattle are equally susceptible to BVD infection, however, both sexes of animals can be infected with BVD at the same or different periods of life, depending on a variety of environmental and management conditions.These factors include the degree of virus exposure and stress exposure.
Regarding age susceptibility, age groups >3-5 years had the mathematically highest rate of BVD infection.This result corroborated those of Daves et al. (2016), who demonstrated a greater prevalence of BVD in adult cattle than in young calves.Our finding might be explained by the fact that animals' risk of contracting BVD increases with age (Daves et al., 2016).Although there was no discernible difference in the rate of BVD infection between native and mixed breeds in the present study, native breeds mathematically had a higher rate than mixed breeds.This result differed from that of Daves et al. (2016), who discovered that cattle of mixed breeds were more likely to be infected with BVD.Our result would suggest that all breeds of cattle are equally susceptible to contracting BVD.Native breeds had higher numerically; since native breeds often have less robust immune systems than mixed breeds.Seasonal fluctuations and the frequency of BVD infection were studied, and found that the percentage of infection with BVD was significantly higher in hot months (26.32%) than in cold months (6.45%).This result was in contrast to that of El-Bagoury et al. (2014), who noted that BVD infection was most prevalent in the winter.Our finding may be attributed to the increased survivability of BVDV at warm temperatures.

CONCLUSION
The current study detected BVD-infected cattle in Assiut governorate, Egypt.These results have economic implications and highlight the necessity of implementing efficient preventative and control measures across Egypt to reduce the prevalence of BVDV.Additionally, to eliminate conditions that exhibit clinical similarities to BVD, such as infectious bovine rhinotracheitis, malignant catarrhal fever, and foot-andmouth disease, all clinical suspicions of BVD must be confirmed by laboratory testing.

Fig. 2 :
Fig. 2: Agarose gel electrophoresis of RT-PCR after amplification of 5'UTR of BVDV in diseased cattle.Line М: DNA ladder 100 bp, line C-ve: Control negative distilled water, line C+ve: Control positive sample, and lines 1,2, 3 and 4: positive serum samples of infected cattle with amplified product at 290 bp.

Table 1 :
Nucleotide sequence of used primers, locations in the 5'UTR of BVDV and size of the products produced after RT-PCR

4.3. Detection of BVDV 5'UTR by RT- PCR
Possibility of a specialized RT-PCR assay for amplifying the highly conserved region of the 5'UTR of BVDV.The extracted RNA

of examined diseased cattle No. of diseased cattle infected with BVD (%)
A: