African swine fever (ASF), caused by African swine fever virus (ASFV), is currently one of the most important and serious diseases of pigs, mainly due to the enormous sanitary and socio-economic consequences. It leads to serious economic losses, not only because of the near 100% mortality rate, but also through the prohibitions of pork exports it triggers. Currently neither vaccines nor safe and effective chemotherapeutic agents are available against ASFV. The disease is controlled by culling infected pigs and maintaining high biosecurity standards, which principally relies on disinfection. Some countries have approved and/or authorised a list of biocides effective against this virus. This article is focused on the characteristics of chemical substances present in the most popular disinfectants of potential use against ASFV. Despite some of them being approved and tested, it seems necessary to perform tests directly on ASFV to ensure maximum effectiveness of the disinfectants in preventing the spread of ASF in the future.
A sensitive and reliable method has been developed and validated to determine residues of abamectin, doramectin, eprinomectin, ivermectin, and moxidectin in bovine milk. Isolation of the analytes from milk was performed with the use of liquidliquid extraction with acetonitrile in the presence of sodium chloride. The extract was defatted with hexane and cleaned up using solid phase extraction (C8 cartridge) after forming ion pairs with triethylamine. The analytes were derivatized with N, Ndimethylformamide, acetic acid anhydride, and N-methylimidazole (100°C, 90 min). The derivatives were determined by reverse phase liquid chromatography with fluorescence detection (excitation and emission wavelength 365 nm and 475 nm, respectively). Recoveries of the lactones from milk samples fortified at 10-30 μg kg-1 ranged from 52% to 80% with intra-laboratory reproducibility (CV) of 12.7%-22.8%. The critical concentrations (decision limit, CCα and detection capability, CCβ) were in accordance with target limits. The method has been verified in the proficiency studies by EURL/CVL Berlin (all z-scores in the range of ±2). The method was transferred to routine laboratories, verified in inter-laboratory comparison and successfully applied in the National Residue Control Plan.
A multiresidue method (LC-MS/MS) for determination of wide range of anthelmintics was developed. The method covered benzimidazoles: albendazole (and metabolites), cambendazole, fenbendazol (and metabolites), flubendazole (and metabolites), mebendazole (and metabolites), oxibendazole, thiabendazole (and metabolites), triclabendazole (and metabolites); macrocyclic lactones: abamectin, doramectin, emamectin, eprinomectin, ivermectin, moxidectin; salicylanilides: closantel, ioxynil, nitroxynil, oxyclosamide, niclosamide, rafoxanid and others: clorsulon, derquantel, imidocarb, monepantel (and metabolites), morantel, praziquantel, and pyrantel. The method was used to examine the potential presence of anthelmintics in goat and sheep milk and dairy products from the Polish market. A total of 120 samples of milk, yoghurt, cottage cheese, cream cheese, and curd were analysed. None of the samples were found positive above CCα (1-10 μg/kg) except for one cottage cheese in which traces of albendazole sulfone were detected (5.2 ug/kg) and confirmed. The results of the study showed negligible anthelmintic residues in the goat and sheep milk and dairy products and confirm their good quality.
African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus’ mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain.
Material and Methods
The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells.
The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate.
Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.