This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly-histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30-IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30-IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30-IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30-IB allowed the detection of ASFV-specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8 days post-infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south-east Africa).
The agent of African swine fever (ASF) is a large envelope virus (ASFV) belonging to family Asfarviridae and containing a double-stranded linear DNA of 170 to 190 kb in size coding for more than 150 proteins, most of which are involved in host-virus interactions (L.K. Dixon et al., 2004). Its virulent isolates cause a contagious hemorrhagic disease with 100 % mortality both among domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa). The disease control is complicated by the lack of any specific preventive methods (R.J. Rowlands et al., 2008; D.A. Chapman et al., 2011; P. Rahimi et al., 2010). The attempts to protect pigs against ASF with experimental live and inactivated subunit vaccines developed by standard methods failed (S. Blome et al., 2014). This paper discusses immunological mechanisms to provide the specific defense base on potentially protective virus-specific proteins, and immunogenic and some protective properties of ASFV gene-based DNA constructs. Immune protection at ASF is due to cytotoxic T-lymphocytes (CTL) and antibody-dependent cellmediated cytotoxicity (ADCC) effectors against viral proteins located on infected monocyte/macrophage. There is a synergism of these effectors (A.D. Sereda, 2013). Based on i) the location, structure and functional properties of viral proteins, ii) the polypeptide specificity of blood antibodies after injecting pigs with ASFV attenuated or virulent strains, iii) the effects of pig immunization using purified proteins from infected cells or the recombinant proteins, and DNN constructs, p30, p54 and CD2v proteins are considered as potentially protective (S.D. Kollnberger et al., 2002; M.G. Barderas et al., 2001; J.G. Neilan et al., 2004). A significant disadvantage of the candidate DNA vaccine is a relatively low immune response, especially in large mammals. There were attempts of overcoming the problem using various strategies (
African swine fever (ASF) caused by African swine fever virus (ASFV) of Asfivirus genus, Asfarviridae family, can occur in peracute, acute, subacute, chronic or asymptomatic form. At early stages of epizootics, the infection usually occurs in its acute form eventually becoming chronic and/or asymptomatic. Seven to ten days post infection the survived pigs develop virus-specific antibodies which persist for a long time. An assumption is reasonable that in the near future, due to repeated passaging ASFV in wild boar populations in European countries, the ASFV isolates may appear which will cause chronic or asymptomatic rather than the acute forms of the disease. In our study we compared different tests to find those the most effective to reveal latent carriers when no apparent symptoms of the disease observed. Thus, our research was aimed at investigation of some special aspects of the laboratory diagnostics of chronic and asymptomatic forms of ASF. The chronic form of the disease was observed in a pig experimentally inoculated with an attenuated ASF virus Stavropol 01/08 A 4 S 2 /9k (at passage 33) at a dose of 10 6.0 HAU 50. On day 5 to 7 post inoculation the signs typical of chronic forms of the infection were registered including depression and fever up to 40.5 С. The antiviral antibody was detected in the swine blood serum from day 7. After the animal was killed on day 21, a haemadsorption assay revealed ASF virus present at low titers in spleen and mandibular lymph node samples while in liver and lung samples it was not found. Based on the results of polymerase chain reaction (PCR), the viral DNA was determined in the mandibular lymph node sample only. Furthermore, immunoblotting assay identified ASF antibody titers of 1:20 to 1:160 in all the organs examined. The asymptomatic forms of ASF were observed in a wild boar yearling which has been intramuscularly inoculated with an attenuated ASF virus strain MK-200 at a dose of 10 7.0 HAU 50. The antiviral antibody was observed in the wild boar serum from day 8. After the animal was killed on day 25, no pathological signs typical of ASF were found, nor was ASF virus found in the organ samples examined using haemadsorption assay or its DNA was detected in PCR. In immunoblotting assay, virus-specific antibodies were identified in liver, spleen, lung and mandibular lymph node samples at dilutions of 1:40 to 1:320. The opportunity of detecting antibodies in spleen, lung and/or liver samples facilitates the monitoring for ASF to be carried out under the infection control campaigns, especially with respect to wild boars shot in game husbandries. Animals sequentially infected with an ASFV low-virulent isolate and a virulent one can survive, in which case it is quite possible to diagnose the disease using both PCR and serological methods. For making laboratory diagnosis of ASF chronic and/or asymptomatic forms, as well as carrying out monitoring studies, serological methods are recommended.
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