Detection of Bovine Ephemeral Fever Virus and Its Effects on Blood Parameters and Serum Calcium Levels in Cattle Population of District Swabi, Pakistan

Among the non-contagious diseases, Bovine ephemeral fever is a disastrous viral disease mainly affecting crossbred cattle population. It is known as “Three Days Sickness” as its course is three days Walker et al., (2012). Cattle and buffaloes are the main species affected by Bovine ephemeral fever (BEF) which gives huge economic losses to the dairy sector due to the sudden drop in milk production. It can be catastrophic for those areas which mostly depend on dairy milk for their livelihood, as drastic losses occur in milk production during infection. The etiologic agent, Bovine ephemeral fever virus belongs to Rhabdoviridae family, enveloped (negative sense) ssRNA virus. It generally recurs in, Asia, Australia, Africa, and the Middle East (Walker, 2005). The transmission of the disease illustrates that it is sporadic in nature and is transmitted by Culicoides spp during heavy rainfall. The virus was isolated from Culicoides spp, collected from the areas where the disease was at its peak Davies et al., (1990). The animals that recover from the disease became immune to the disease and are seldom re-infected. The devastating effects of the disease are that the animals become low productive Davies et al., (1975). The virus is present basically in blood plasma and leukocytes: it is detected during and before the onset of fever (Young and Spradbrow, 1985; Liao et al., 1998; St. George et al., 1984; Finlaison et al., 2014). Clinical signs of the disease are biphasic fever, temporary lameness, stiffness, loss of appetite, muscle fasciculation, nasal and ocular discharges, swelling of joints, paralysis of limbs, anorexia, profuse salivation, ruminal stasis, depression, and recumbency. The disease mostly flourishes in lactating or healthy animals. Infected animals mostly recover after three days (Kirkland, 2002).
        
Swabi is one of the densely populated districts of Khyber Pakhtunkhwa, Pakistan, and its fertile land provides a good opportunity for the rearing of livestock. Different cattle breeds are reared by farmers, mainly for milk. There are no studies reported on bovine ephemeral fever (BEF) in Khyber Pakhtunkhwa province, Swabi district of Pakistan. BEF is a big hurdle to the dairy sector in Swabi district due to the lack of proper diagnosis and awareness of the disease. Therefore, the present study was conducted to identify the causative agent of bovine ephemeral fever through nested Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), to confirm the diagnosis of diseased cattle suspected of three days sickness. Hematological analysis and serum calcium level profile were studied in confirmed cases to see the effects of infection. In addition, this study provides basic information on BEF which can be helpful in providing the control strategies of the disease in the area.

Samples were collected in the months of July and August 2014 from private, public sector dairy farms, Khushbakht Dairy Farm, Taif dairy farm, Khalid dairy farm, Naeem Yousafzai dairy farm, Ibraheem Dairy farm, Gadoon dairy farm and from individual animal keepers in Swabi district. The studied animals were cattle suspected of bovine ephemeral fever of all age groups and breeds showing the clinical signs of a high fever (41-42°C), lameness, hypocalcemia, nasal and ocular discharges, muscle fasciculation, swelling of joints, paralysis of limbs, severe decrease in milk production, and recumbence. A total of 50 blood samples were collected from the suspected animals under aseptic condition using a sterilized disposable syringe and were preserved in vacutainers (Anticoagulant added n = 50, without anticoagulant n = 50). The blood samples from healthy cattle (n=10) were collected as experimental control: (Anticoagulant added n = 10, without anticoagulant n = 10), for comparison between infected and healthy animals.
 
Reverse Transcriptase-Polymerase Chain Reaction
 
Buffy coat was separated using centrifugation at 6000 RPM for 10-15 minutes Hughes et al., (2012). Following centrifugation, three layers were observed (the lower layer contains RBCs, the middle layer consist of a buffy coat and the upper layer was plasma). Carefully aspirate the buffy coat using a dropper. The RBCs were lysed with distill water followed by addition of 1X phosphate buffer saline (PBS). The buffy coat cells were used for RNA extraction. TRIzol reagent (Invitrogen, Carlsbad, CA USA) was used for extraction of total RNA from the solutions, adopting the methodology described by the manufacturer Rio et al., (2010). A reverse transcription (RT) reaction was carried out using a cDNA synthesis kit (#K 1622 Thermo Scientific Revert Aid First Strand cDNA Synthesis kit, USA) adopting the manufacturer’s instructions Jung et al., (2013).
        
The conserved G gene was amplified by Polymerase Chain Reaction from all samples using Dream Taq Master Mix (2X) (#K 1071 Thermo Scientific, USA) with two steps Polymerase Chain Reaction using the two primers sets given below (Bakhshesh and Abdollahi, 2015).
 
Reaction 1: The live vaccine of bovine ephemeral fever manufactured by Choojg Ang Vaccine Laboratories Co., Ltd. 59-3 Hwaam-dong, Yuseong-gu, Daejeon, the Republic of Korea was used as a control positive. PCR amplification was carried out by using the following temperature and time: Initial denaturation temperature was 94°C for 5 minutes followed by denaturation of 94°C for 30 seconds, annealing at 56°C for 1 minute, Extension at 72°C for 1 minute, followed by 35 cycles and a final extension at 72°C for 10 minutes (Bakhshesh and Abdollahi, 2015).
 
Reaction 1: (position 346–369 and 1155–1132)
BEF346F 5'-TATTACCCTCCTGCCGGATGCTTT-3'
BEF1155R 5'-AGGTCTGTATTCGCACCAAGCTCT-3'
 
Reaction 2: Reaction 2 was performed on the product of the first reaction by taking 1 µl product of the first reaction diluting it 1/100 µl and then took the 1 µl of it as a template DNA for 2^nd reaction. Thermal conditions and cycles were the same except annealing temperature at 54°C. The PCR results were recorded using 2% agarose gel by Gel Electrophoresis.
 
Reaction 2: (position 79–104 and 584–560)
BEFGTR08-F79 5'-GGAGAACTTCATCCTGTAAAGGCCCA-3'
BEFGTR08-R584 5'-ACTTCTGGTATCCATGTCCCGGTT-3'
 
Hematological analysis
 
About 3 ml of blood was collected in a 5 ml syringe from the suspected and healthy cattle for the comparison of BEF suspected and healthy cattle. Every blood sample was transferred in tubes of EDTA purple top tubes for determination of hematological parameters. Blood samples were used to determine the complete blood count (CBC) through automatic hematology analyzer (Abacus, Diatron Messtehnik Ges.M.B.H. Wein, Austria) Becker et al., (2008). The estimated hematological parameters included: total erythrocyte count (RBC), packed cell volume (PCV), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cells (WBC) neutrophils lymphocytes and MID (it include monocyte, basophils and eosinophils).
 
Serum calcium
 
For serum calcium, a total of 3 ml blood was collected in plain tubes without anti-coagulant from the jugular vein of sick cattle. The confirmed positive samples (confirmed by RT-PCR) and 10 healthy samples (confirmed by RT-PCR) were analyzed by chemistry analyzer for the changes in serum calcium level at the University of veterinary and animal sciences, Lahore Pakistan Higbie et al., (2015).
 
Statistical analysis
 
The data were analyzed for comparison between healthy and confirmed positive samples for BEF (Bovine ephemeral fever) under hematological analysis and calcium levels using Two Independent sample T-test.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Bovine ephemeral fever virus was detected in 33 out of 50 samples in suspected cattle with the aid of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). It was also evaluated in uninfected healthy cattle but the virus was not diagnosed in uninfected or healthy cattle. Results of PCR identification showed 66% positive animal of bovine ephemeral fever virus. Nested RT-PCR was applied to all 50 samples. In the first reaction 809 bp RT-PCR product was obtained by Agarose gel electrophoresis (Fig 1), while in the second reaction it was 505 bp RT-PCR (Fig 2).
 

 

 
Hematological indices in bovine ephemeral fever virus infected and healthy cattle
 
Blood samples of 50 suspected cattle for BEF (salivation, high temperature, lameness, anorexia and decrease in milk production was observed) were taken, 33 out of 50 samples were confirmed through RT-PCR. These 33 positive samples for BEFV were compared with 10 healthy cattle samples, and there was a significant increase (p < 0.05) in neutrophils number of infected cattle as compared to healthy cattle. Neutrophils 10.44 ± 1.87 × 10⁹/L was recorded in infected cattle, while there was a significant decrease (p < 0.05) in lymphocyte which was 2.90 ± 0.97 × 10⁹/L as compared to healthy animals. No significant change (p > 0.05) was observed in red blood cells (RBCs), hemoglobin (Hb), pack cell volume (PCV), mean corpuscular volume (MCV), mean concentration of hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs) and MID (Table 1) (it includes the monocyte, eosinophils and basophils).
 

 
The Mean value of Serum Calcium in BEFV infected and healthy cattle
 
Serum samples of 50 suspected cattle for BEF were taken, 33 out of 50 samples were confirmed positive through RT-PCR. These 33 positive samples for BEFV were compared with 10 healthy cattle samples for the serum calcium level. The serum calcium level of confirmed BEF positive samples was recorded as 7.84 ±0.16 mg/dL and there was significant decrease (p < 0.05) in serum calcium level while the serum calcium level of the 10 healthy cattle was recorded as 8.75 ± 0.45 mg/dL (Table 2).
 

        
BEF is one of the important viral infectious diseases of cattle and buffalo in Pakistan (Roya, 2008). In July 2014, BEF was noticed in Pakistan. Here, we confirm the diagnosis of BEF in suspected cattle using RT-PCR and reported changes in hematological profile and serum calcium level in infected cattle. The hematological results showed the involvement of the hematopoietic system which was evident from the overall increase in neutrophils and decrease in lymphocytes, this drop is associated with the production of cross-neutralization antibodies Gray et al., (2011). No changes were observed in erythrocytes count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume and MID (it includes eosinophils, basophils and monocytes). A Significant decrease in serum calcium level was also observed.
        
In this study, we detected BEFV through RT-PCR in animals suspected to be infected with BEF and confirm infection in 33 (66%) out of 50. Similar methods were applied in previous studies, where 60 blood samples were collected from suspected cattle, virus isolation was done by culturing the samples in Vero cell line (green monkey kidney continuous cell line) and BHK-21 (baby hamster kidney continuous cell line). These samples were confirmed by RT-PCR and 66.67% (40 out of 60 cows) cases were positive (Zaher and Ahmed, 2011). Accordingly, Finlaison et al., (2014) reported a 69.4% infection prevalence by RT-PCR. Although, Degheidy et al., (2011) reported a lower infection rate in suspected cattle when compared with our result, but with a different approach. In their study, buffy coat were separated from suspected blood, followed by virus intra-cerebral inoculation in baby mice and in tissue culture and reported a 23.1% (22 out of 95 suspected samples) and 17.8% (17 out of 95 suspected samples) from direct collection and mice inoculation, respectively unlike in our study where RNA was extracted directly from suspected blood samples, followed by cDNA production and amplification of the G gene by RT-PCR. Since, BEF and other diseases such as babesiosis, black quarter, acute laminitis present similar clinical conditions, the difference in prevalence may have resulted in the collection of samples from suspected animals with the aforementioned other than BEF. Also, the geo-climatic conditions of both the areas may account for the differences.
        
The increased level of neutrophils during the peak of the temperature may be due to the bone marrow response against the virus and presence of increased number of band neutrophils. Contrastingly, we observed a significant decrease in lymphocytes levels, which has been previously reported, this decrease is believed to be inversely related to neutrophil number (St George et al., 1984; Uren and Murphy, 1985).
        
In accordance with the report of St George et al., (1984), we observed a significant decrease in serum calcium level of infected cattle and marked hypocalcaemia which was less than previous report of 2 mmol/L Uren et al., (1992) and closely similar to reports by (Thabet et al., 2011; Uren and Murphy, 1985). A reason for hypocalcaemia during ephemeral virus infection has been linked to activated neutrophils Leno et al., (2017). During neutrophil activation, the calcium in the plasma migrates to the interior of the neutrophil causing hypocalcaemia. The activated neutrophils decrease the calcium level. This level normalizes at the end of the lifespan of the neutrophils Valli et al., (1971). Muscle fasciculation was also observed and this is due to the low calcium levels. Other possible reasons may be: ruminal stasis, increase pH induced by high ammonia levels, and presence of some non-esterified fatty acid in the blood plasma Murphy et al., (1990).

This study shows the advantage of rapid diagnosis of BEF from buffy coat. The advantage of the buffy coat is the reduction in detection time when compared to previously reported, time-consuming RT-PCR approach. Furthermore, we observed the relationship between BEFV infection and the increase neutrophil level, decreased lymphocyte number and a marked decrease in serum calcium level. This is the first molecular detection performed on BEF in Pakistan. To this end, the data generated from this study will provide the basic information on Bovine Ephemeral Fever in the selected area and will help to formulate strategies for control of disease in the area.