Investigation of The Association Between Bovine Viral Diarrhea Virus and Neospora caninum as a Cause of Abortion in Cattle

This study aimed to investigate the complementary association between Bovine Viral Diarrhea Virus (BVDV) and Neospora caninum (NC) in the induction of abortion in cattle. The study was carried out on 116 serum samples collected from 116 aborted cows in different Egyptian localities. All blood samples were immediately transported to the diagnostic laboratory. Serum was harvested after centrifugation at 1500×g for 10 min. All sera were divided equally into two microtubes and stored at -20 °C until laboratory testing. Double antibody sandwich immunoenzymatic assay (DS.ELISA) is used for bovine viral diarrhea virus (BVDV) p80/p125 antigen detection and Anti-Neospora caninum (NC) antibodies were detected using an indirect ELISA. Our results cleared that the incidences of BVDV and NC among examined cow's serum samples were 66 (56.9 %), where the incidences of BVDV and NC were 31 (26.72 %) and 35 (30.17 %), respectively. There were 18 (27.3 %) samples that showed mixed infection BVDV and NC incidences among examined cattle serum samples. The study concluded that the prevalence of BVDV and NC are high and widely spread in cattle farms in Egypt. There is a pronounced association between BVDV and NC and concurrent infection in cattle farms in Egypt. So we should coordinate surveillance and control programs on abortifacient pathogens (BVDV and NC) should be developed and implemented, which would reduce the losses associated with these diseases in cattle farms. ـــ ــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Abortion is critical to the dairy sector because it hinders heifers and cows' scheduled lactation and replacement. It also leads to culling, which has a negative impact on herd genetics, resulting in lower farm revenue. Furthermore, abortion storms can be psychologically devastating to the farmer. Many diseases cause cattle abortion as Campylobacteriosis, Leptospirosis, andBrucellosis (Carpenter et al., 2006 andOlmo et al., 2018). However, there is a scarcity of research on other additional diseases such as bovine viral diarrhea (BVD) and neosporosis as well as their interrelationships as causes of abortion. Nonetheless, these play a significant influence in reproductive wastage globally (Okumu, 2014 andOkumu et al., 2019).
Bovine viral diarrhea is considered one of cattle's most important viral diseases worldwide (Yitagesu et al., 2021). This is a Pestivirus in the family Flaviviridae closely related to the border disease virus of sheep and the classical swine fever virus of pigs (OIE, 2018). The virus has a wide distribution, and certain animals may have subclinical infections (Lindberg and Houe, 2005).   Silva et al., 2007). Neospora caninum causes abortions in calves as early as 3 months of pregnancy; however, they are most common around 5 and 6 months. One of the obvious evidence of infection of cattle is foetal resorption, autolysis, mummification, and stillbirth; however, some calves are born alive with neuromuscular problems and others appear healthy with persistently infected (Dubey and Schares, 2006).
Congenital/vertical transmission of neosporosis from seropositive dams to their offspring is prevalent, and abortion is common in future pregnancies (Dubey et al., 2007). Reported risk factors for bovine abortions due to Neospora caninum include geographical location, exposure to dogs, and being pregnant heifers (Dubey and Schares, 2006; Koiwai et al., 2006).
Neospora caninum is an intracellular protozoan parasite that is found globally and is a serious economic concern in the cattle sector. According to Neospora caninum and bovine viral diarrhea virus have been reported as major causes of bovine abortion worldwide (Olum et al., 2020). There has been a hypothesis that both diseases have a synergism in inducing abortion, while there's no evidence to back this up (Konnai et al., 2008). This study aimed to investigate the association between BVDV and Neospora caninum infection as causes of abortion in cattle.

Animals:
One hundred sixteen aborted cattle from 15 small to medium-sized cattle farms in different localities (Giza, El Fayoum and Beni Suef governorates). All the farms were vaccinated only with routine vaccination. The animals have a history of abortion. The abortion percentage in these farms ranged between 6 to 14%. The aborted animals were from 3 to 8 years old. Most aborted cases were in the first trimester and the rest were in the second trimester.

Sampling:
One hundred sixteen serum samples were collected from 116 aborted cattle. Blood samples were collected by jugular venipuncture followed by centrifugation at 1500 ×g for 10 min. All sera were divided equally into two microtubes and stored at -20 °C until testing in the laboratory.

Neospora Caninum Indirect ELISA:
All 116 Serum samples were analyzed for anti-Neospora antibodies were detected using an indirect ELISA ID. Vet kit (France). The manufacture instructions were followed when using kits. Read in an ELISA microplate reader at a wavelength of 450 nm. For each sample, calculate the S/P percentage (S/P %): The sample (OD sample ) is divided by the mean positive control value (OD PC ) multiplied by 100.

BVDV Sandwich ELISA:
All 116 sera samples (50μl) were submitted to BVDV antigen ELISA (ELISA BVDV-Ag) using (INGEZIM BVD DAS) kits. All procedures were performed according to manufacturing instructions. Briefly, ELISAs are carried out in solid-phase microplates. Wells are coated with anti-BVDV antibodies. Samples to be tested (serum) and controls are added to microwells. Anti-p80/p125 Biotin conjugated monoclonal antibodies (MAbs; conj.1) is added and subsequently, Streptavidinperoxidase conjugate (conj.2) is added to each well. After washing, the substrate solution (TMB) is added and the optical density (OD) is read at 450 nm. The tests can be considered valid if the ratios OD of the positive control/OD negative control ˃ 10.

Results interpretation:
Cut off = OD of positive control x 0.1 Samples with an OD higher than the cut-off value plus 15% must be considered as positive. Samples with OD lower than the cut-off value minus 15% must be considered as negative. Samples with OD values between both values (of positive and negative samples) are considered as doubtful. S/P ratio was ≥0.2

Statistical analysis:
The statistical analysis was carried out using the Chi-square test to examine the incidences of BVD, NC and Both BVD with NC among examined cattle serum samples using SPSS PC+ -Version 20.

RESULTS
The result showed that 66 of examined animals (N=116) were positive to BVDV and/or NC 66 (56.9%), where the incidences of BVDV and NC were 31(26.72%) and 35(30.17%), respectively. There were 18 (27.3%) samples that showed concurrent infection BVDV & NC incidences among positive cow serum samples as shown in Fig (1).

Incidence of bovine viral diarrhea virus:
Results cleared that, the incidences of BVDV among examined cattle serum samples was 31(26.72 %) and was (47%) among +ve samples as in table (1).

Examination of the prevalence of BVD, NC, and concurrent infections in the serum samples of the cattle examined using Chi square test.
All the percentages related to the total number of samples (116) showed a high but non-significant relationship between BVDV and NC (Table 4) .

DISCUSSION
The detection of Bovine Viral Diarrhea in the present study was done using a double-antibody sandwich immunoenzymatic assay that detects BVDV p80/p125 antigen. As high conserved immunogenic antigen in cytopathic (p80) or noncytopathic (p125) biotypes, it is well detected in PI animals' blood sera. Because of that, this test has formed the basis for successful eradication policies in many countries as a more rapid screening test and generating information on the infectious status. Also, Ag-ELISA showed a high sensitivity for detecting BVDV infected calves (Cornish et al., 2005).
Our results cleared that, the incidences of BVDV among examined cattle serum samples was 31(26.72 %). It was (47%) among positive samples Table (1); these results were agreed with those of (El-Bagoury et al., 2012), where they reported that the seroprevalence of BVD was 40% and 23% for cattle and buffaloes, respectively. Herd biosecurity strategies like disinfection, double fencing, and keeping a closed herd have been reported to diminish the prevalence of BVDV infection. (Kampa et al., 2004: Gates et al.,  2013). BVDV infection of pregnant cows and heifers has been recorded as a cause of abortion and other reproductive abnormalities, as well as increased age at first calving and decreased ovarian function in affected herds, which could explain the widespread BVD infections and associated economic losses. (Kabongo and Van Vuuren, 2004; Heuer et. al., 2007). Bovine Viral Diarrhea Virus has also been reported to be fetopathogenic in cattle, thus leading to early embryonic death, repeat breeder syndrome, and abortion (Yang et al., 2012 and Yitagesu et al., 2021). (Olum et al., 2020). Moreover, animals that are both serologically positive for Neospora spp. and positive for BVDV antigen in viral isolation or ELISA-Ag tests are thought to be co-infected by the two agents Our results showed that the incidence of NC among examined cattle serum samples was 35 (30.17 %). In comparison, it was (53%) among positive samples Table (2), these results were agreed with that recorded by (Dubey et al., 1998) where the prevalence of N. caninum antibodies were 68% and 20.43% in buffalo and cattle, respectively in the delta region in Egypt. Our results are higher than that reported by (Fereig et al., 2016), where they revealed that 18.9% of examined cattle were positive for N. caninum. Also, NC prevalence was high compared to other studies that had reported prevalence ranging from 1.9%-39.7% (Konnai et al., 2008; Asmare et al., 2013). This may be due to the low level of knowledge by farmers and animal health providers on abortion caused by animal health. Thus, the high prevalence of NC in this study may be associated with the low level of knowledge on this disease and the subsequent lack of a control program (Sarrazin et al., 2013), in addition to the role of stray dogs in Egyptian cattle farms.

It was proved that all animals which have antibodies to Neospora spp are diagnosed as persistently infected (Bachofen et al., 2010) While the detection of BVDV virion or viral antigens indicates both acute infection (transiently infected animals -TI) and persistent infection (persistently infected animals -PI)
On other prospects, our results agreed with the results of (Fereig et al., 2016), where they reported a higher incidence of infection on small cattle farms compared with large farms; the incidences were 50.2% versus 37.8%, respectively. They observed that cows in Sohag had a considerably greater prevalence than that in Qena, with a seroprevalence of 46.1% compared with 31.6%, respectively. Pathogenesis of NC leads to abortions and fetal losses by inducing placental damage or causes the release of maternal prostaglandins that in turn cause luteolysis and abortion (Dubey et al., 2007  and Olum et al., 2020). The high seroprevalence recorded in this study indicates a high incidence of NC infections in cattle.These necessitates the application of more effective strategies for combating these types of infections on farms in Egypt.
Our results observed that, the concurrent incidences of BVDV with NC among examined cows serum samples was 18 (27.3%) Table (3), this gives a clear illustration about the association between BVDV and Neospora caninum, that play role or may be involved in the induction of abortion in cattle where the percentages of concurrent infection were (15.52%) among total examined samples. The statistical examination of BVD, NC, and concurrent BVD and NC incidences among studied cattle serum samples showed a high but non-significant relationship between BVDV and NC, Table (4). These results agreed with (Quinn et al., 2004 andLassen et al., 2012). They found a non-significant correlation between BVDV infection and Neospora spp. On the contrary, (Duong et al., 2008) found a significant association between N. caninum and BVDV in small cattle farms. The high incidence rates of the NC and BVD was observed in their study (12.9%); the most common cause of fetal loss was Neospora caninum (29.0%), followed by mixed NC and BVDV infections of NC and BVDV (12.9%), BVDV (9.9%).
In addition, the 102 dairy cattle seropositive to   (Yitagesu et al., 2021). NC might be a secondary cause of abortion and also may exacerbate abortion caused by other agents (Asmare et al., 2013).
The high level of seropositivity in this study may have been due to the lack of adequate control measures such as vaccination, screening and isolation of new introductions and improved biosecurity for these two diseases; this was probably due to lack of awareness of the presence of the conditions in dairy cattle in Egypt and consequently low levels of knowledge by the animal health industry players on the impact of these diseases

CONCLUSION
This study concluded that, the prevalence of BVDV and NC are high and widely spread in a cattle farm in Egypt. There is a high non-significant association between BVDV and NC and the concurrent infection present in more than a quarter of positive cases. So we should coordinate surveillance and a comprehensive policy on the control BVD and NC should be developed and implemented by the government with the involvement of all stakeholders in the dairy industry; laboratories should offer diagnostic tests for BVDV and NC to help farmers determine their roles in controlling abortions on their farms.

Declaration of Conflicting Interests
The authors revealed that there is no potential conflicts of interest.