Comparison of African Swine Fever Virus Prevalence in Nigerian Indigenous Pig, Its Hybrid and Backcross in an Environment Prone to African Swine Fever Abstract This Work Investigated on the Presence of African Swine Fever Virus (asfv) in Nigerian Indigenous Pig (nip), Its Hybrid and Backcross Using 

amplified the 278 bp of ASFV in the DNA extracted from NIP, its hybrids and backcross. In conclusion , this study has shown that NIP, its hybrid and backcross have ASFV in their genome in an ASF-prone environment and thus confirming the continuous prevalence of ASF in Southwest Ni-geria. This is an ongoing research where the severity and virulence of the virus has to be measured .


Introduction
Pigs play a major role in socioeconomic life of the people; it serves as a source of income for rural population and fulfill important role in culture and food security.The population of pig in Nigeria increases from 2 million in 1984 to 7 million in 1997 before the widespread of ASF epizootic [1].
Disease is one of the factors that affect the livestock production in Nigeria [2].Jovanoic et al., 2009 [3] stated that diseases can have a significant impact on animal productivity and production, human health and, consequently, on the overall process of economic development Pigs harbor a range of parasites and diseases some of which are zoonotic.One of these is the African Swine Fever that is caused by a virus.
African swine fever (ASF) is a highly contagious viral disease of pigs and of such concern that it is included among the List A diseases by the United Nations Office International des Epizooties (OIE) [4]- [8].It causes a devastating haemorrhagic fever of pigs with mortality rates approaching 100% with the acute and peracute forms.It causes major economic losses, threatens food security and limits pig production in affected countries.The disease causes significant economic losses in affected countries due to the high mortality rates associated.The transmission of the disease, as it now occurs in sub-Saharan Africa, is through the African soft tick (Ornithodoros moubata porcinus) and Warthogs or domestic pigs.The transmission through the warthog and soft ticks does not occur in West Africa, although ASF virus has been detected from Warthog in Nigeria of West Africa [9] and also in local pigs in Nigeria [10].The presence of soft ticks is also confirmed in Warthogs [7].
The diagnosis of infectious diseases such as ASF can be performed by direct and/or indirect detection of infectious agents.By direct methods, the particles of the agents and/or their components, such as nucleic acids, structural or non-structural proteins, enzymes, etc., are detected.The indirect methods demonstrate the antibodies induced by the infections [11].The most common direct detection methods are isolation or in-vitro cultivation, electron microscopy, immunofluorescence, immunohistochemistry, antigen enzyme-linked immunosorbent assay (antigen-ELISA), nucleic-acid hybridisation (NAH), macro-and microarrays and the various techniques of nucleic acid amplification, such as the polymerase chain reaction (PCR) or the isothermal amplification methods, such as Nucleic Acid Sequence Based Amplification (NASBA), Invader or Loop-Mediated Isothermal Amplification (LAMP).This study aimed at comparing the ASFV prevalence in NIP, its hybrid and backcross in an environment prone to African Swine Fever.

Blood Sampling
Blood samples were collected from the jugular vein of pure NIP (20), crossbreds (10) and Backcross [14] into 5 ml sterile EDTA bottles and brought into the laboratory for haematological analyses and DNA extraction for Polymerase Chain Reaction (PCR).

DNA Extraction
The DNA extraction was done by using DNA kit (Zymobeads).The manufacturer instructions were followed in details.

PCR Condition
Diagnostic Polymerase Chain Reaction was performed according to the Manual of Diagnostic Tests and Vacc-ines (OIE, 2008).ASF-specific primers (oligonucleotide primers) targeting the major capsid protein (VP72 gene) amplifying a 278-bp fragment within the conserved region was employed: PAS 1 F: 5'-ATG GAT ACC GAG GGA ATA GC-3' and PAS 2 R: 5'-CTT ACC GAT GAA AAT GAT AC-3' (Luther et al., 2007;OIE, 2008).The final reaction volume of 25 μl PCR master mix comprised 10 μl extracted DNA template, 10 μl Nuclease Free Water, 10 μl oligonucleotide primers (for both Forward and Reverse) and 12 μl of already prepared Master Mix.Each tube was placed in an automated PCR thermal cycler (MG48+; Mygene TM Series) for amplification for 35 cycles as follows: initial denaturation at 94˚C for 3 minutes for 35 cycles, with 3 steps of denaturation at 94˚C for 30 seconds, annealing at 57˚C for 45 seconds and extension at 72˚C for 30 seconds and final extension at 72˚C for 5 minutes.Amplification products were analyzed by electrophoresis on a 1% agarose gel containing 0.5 μg of ethidium bromide per ml.The gel were visualized under Ultra violet light and photographed.

ASFV Detection
PAS Primers of ASFV amplify at 278 bp for infected pigs used as positive control, NIP, hybrid and the crossbred.Plate 1 shows single and discrete band of ASFV amplification at 278 bp for NIP and for a positive control sample obtained from the Veterinary Medicine of the University of Ibadan while no band was observed for the negative control.
The Pearson correlation tables for haematological traits of Hybrid, NIP and backcross respectively are presented in Tables 3-5.There was high positive correlation (1.00) between Packed Cell Volume and haemoglobin in all the three breeds; and there was negative correlation (−0.99, −0.97 and −0.96) between Neutrophil and the Lymphocyte of Hybrid, Backcross and NIP respectively.This negative correlation between the Neutrophil and Lymphocyte mean that increase in Neutrophil will cause decrease in Lymphocyte.

ASFV Amplification for NIP, Hybrid and Backcross
The 278 bp of ASFV observed in NIP can be corroborated with the findings of Luther et al. (2007) [9] and Luther et al. (2008) [13], where the same virus band was observed in Bushpigs and Warthogs tested at National Veterinary Research Institute (NVRI), Jos.The presence of ASFV in NIP for long period without any clinical symptom or death was explained by Adeoye and Adebambo (2010) [14] and Oluwole and Omitogun (2013) [10] by the ability of the pigs to trap ASFV within their tissues by the activities of macrophages that eat up the pathogens and infected tissues so that other parts and their mast cells in the tissues are not affected.The Hybrid and backcross also showed the presence of ASFV.This result was corroborated with the findings of Adeoye and Adebambo (2010) [14], where serological tests showed the presence of ASFV in crossbreds.The hybrid and the backcross showing the presence of ASFV was not surprising as ASFV were present in the NIP parent.This observation in hybrid and backcross was reported in their work where serological tests were carried out on ASF outbreak survivors, their offspring and F 2 showed a decline in antibody levels against ASF from 100% to 18.79%.This phenomenon was explained as the ability of engulfed ASFV to be broken down easily and effectively by macrophages leading to decreasing circulation of ASFV in the blood and other tissues.Thus, the amount of shed ASFV particles observed in urine and feaces was seen to drop significantly in the offspring.This observation was also corroborated by Olugasa et al. (2007) [15] who also reported that the level of infection in the serum dropped from parents with 96.8% to 13.8% in their offspring.
The ability of the virus to persist in one host while killing another genetically related host was established by Palgrave et al. (2011) [16] where a particular sequence found in warthog and bush pigs was absent in domestic pigs.

Leukocyte Traits in NIP, Hybrid and Backcross
The White Blood Cell (WBC) count is a powerful indicator for infectious and inflammatory disease, leukemia, lymphoma and bone marrow disorders.WBC was found to be higher in backcross compared with NIP and highest in hybrid (Table 1).WBC was high in Hybrid because it increased to fight against the virus.Thus, the Leukocyte traits obtained from this study were indications of NIP and backcross been exposed to ASFV and the susceptibility of hybrid to the virus.WBC of NIP (18.16 ± 3.01 × 10 3 /µl) was within the normal range (7 to 20 × 10 3 /µl) for domestic pigs while WBC of hybrid and backcross values of 31.27 ± 1.79 × 10 3 /µl and 27.71 ± 2.01 × 10 3 /µl were higher than the normal range (7 to 20 × 10 3 /µl) (Table A1 in Appendix).This deviation from normal range for domestic pig was also observed to be in agreement with Karalyan (2012) [17], Anderson (1998) [18] and Eze et al. (2010) [19] where the WBC (25.92 ± 8.08 × 10 3 /μl) of the pigs from South-East of Nigeria were higher than the normal or physiological values (7 to 20 × 10 3 /µl).The observed increase in WBC of Hybrid and backcross was adduced to a probable virus inducement of mass-scale mortality of lymphocytes and neutrophils causing the presence of additional nucleus as reported by Karalyan (2012) [17] and Anderson (1998) [18].Karalyan (2012) [19] also reported that ASFV infection leads to serious changes in composition of WBC.Stress effects induced due to the presence of pathogen was also presented as a possible cause of increase in WBC [17] [18].The same trend as observed in WBC was observed in neutrophil and Eosinophil of male and female pigs (Table 2).Scientists reported that the neutrophil percentage increased during stress [20].The significance of increase in Eosinophil of females (1.07%) compared to 2.12% in males in Table 2 might be due to stress that may be caused by the ASF diseases.Addah et al. (2007) [21] reported that high Eosinophil is responsible for the stress, and diseases [22].

Red Blood Cell Count in NIP, Hybrid and Backcross
Red blood cell parameters are important innate immune cells in blood circulation and they can recognize antigen, kill antigen, they are also involved in immune regulation and have a complete self-regulation system.The RBC of hybrid, NIP and BC were not significant varied from each other from the study.This finding was contrary to findings of De et al. (2013) [23] where the PCV values of local pigs of India were significantly different from that of the exotic pigs.In this study, values for RBC, PCV and Hb were observed to be within normal range.Karalyan et al. (2012) [17] suggested that ASFV directly or indirectly influenced the haematopoietic homeostasis through activation or impaired haematopoiesis [17] [24].Also Wardley and Wilkinson (1977) [25] and Quintero et al. (1986) [26] reported that ASFV is associated with RBC.Karalyan et al. (2014) [19] reported that from the beginning of infection, juvenile forms of RBC, such as the largest cells were observed in the peripheral blood of infected pigs.Among the erythroid precursors, up to 60% of all cells were binucleated, which indicates that acute ASFV infection is accompanied by the emergence of pathological forms of RBC.
The insignificant effect of sex on MHCH and MCH is also corroborated [27] [28].

Platelet Count in NIP, Hybrid and Backcross
The significant effect of breed on platelet trait was in line with findings of De et al. (2013) [23] where the variation between the local breed from India and exotic pigs were significant.Platelet trait in this study for NIP and Hybrid were within the normal range (but that of Hybrid was higher than that of NIP) while the backcross value was lower than the normal range for the domestic pigs.Platelet trait is one of the inflammation signals.Gomez-Vilamandos et al. (1996) [24] reported that platelets assist in dissemination of ASFV within infected pig body particularly in sub-acute infection while Anderson (1998) [18] reported that platelet count decreases during acute infections of ASF.

Conclusions
In conclusion, ASFV was present in NIP, hybrid and the backcross.The inflammation signal (white blood count, red blood count and platelet count) in hybrid was higher than that of NIP and backcross.ASF in West African countries especially Nigeria, has become endemic because of the continuous occurring of the intermittent infections.This disease causes large fatalities in pigs and consequently significant loss of income and employment opportunities [29].It has an overall effect on the pig industry worldwide, because it limits opportunities to explore external markets and increase rapidity/possibilities of inter-continental contamination.
When the presence of the virus is identified, the focus should be on environmental risks and biosecurity in pig.Fashina [30] reported that 306 pig farms and 91% of the 31,916 affected pigs succumbed to the disease in Ibadan (2001), South west Nigeria.Compulsory slaughtering of infected animals and animals at risk, without any compensation to the owners, has dramatic effect on the piggery farmers.The threat of losing their animals with no recompense, drives pig-keepers to do two things to cut their losses, If possible, they will move their animals away from the infected areas, maybe to relatives in neighboring states or countries.In this part of Africa, where sociocultural ties exist regardless of national boundaries, it is relatively easy to move animals over the border to kinsfolk, thereby causing the spread of the disease.

Table 1 .
Effect of genotype on the haematological parameters of NIP, Hybrid and Backcross.

Table 3 .
Pearson correlation coefficients for haematological traits of NIP.

Table 4 .
Pearson correlation coefficient for haematological traits of hybrid.