Jakov Nisavic

Molecular detection of PCV2 and PPV in pigs in Republic of Srpska, Bosnia and Herzegovina.

Abstract The presence of porcine circovirus 2 and porcine parvovirus was examined in forty clinical samples of spleen, lymph nodes and lungs originating from non-vaccinated swine by polymerase chain reaction. All animals were reared in extensive livestock farming systems in different geographical districts of Republic of Srpska, Bosnia and Herzegovina. Porcine circovirus 2 DNA was detected in four lymph node and two spleen samples (15%), while porcine parvovirus DNA was identified in five lymph node samples (12.5%). The presence of both viruses was detected in three lymph node samples (7.5%). Partial nucleotide sequence of ORF1 gene of 2 porcine circovirus 2 and VP2 gene of 2 porcine parvovirus isolates was determined. The nucleotide sequences of two PCV2 isolates from RS-BIH included in phylogenetic typing are similar and cluster together with the strain Mantova isolated from domestic pigs in Italy, strains DE006-14 and DE222-13 isolated from pigs in Germany as well as with the strain Jvnan isolated from pigs in China. Also, analyzed PCV2 isolates were partially similar to the strain NIV-C SRB isolated from pigs in Serbia. The nucleotide sequences of two PPV isolates that were included in phylogenetic typing showed a high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated from pigs in USA and strains 77 and LZ isolated from pigs in China.

Molecular Detection of Pseudorabies Virus (PrV), Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV2) in Swine in Republic of Montenegro

Abstract The presence of pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) was examined in sixty samples (spleen and lymph nodes) and thirty samples of sacral ganglia collected from non-vaccinated swine by virus isolation and polymerase chain reaction (PCR). Using PCR method PrV was detected in three samples, PPV in seven samples and six samples were found positive for PCV2. The phylogenetic analysis of the nucleotide sequences of three PrV isolates identified in this study showed high similarity and significant clustering within the PrV genotype I strains such as Kaplan and Bartha isolated from pigs in Hungary, strain Becker isolated in USA and strain Kolchis isolated in Greece. The nucleotide sequences of two PPV isolates showed high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated in USA and strains 77 and LZ isolated in China. The phylogenetic analysis of the nucleotide sequences of two PCV2 isolates showed high level of similarity and significant clustering within genotype PCV2b strains such as NIVS-3, NIVS-5 and NIVS-6 isolated in Serbia, strain 3959 isolated in Austria, strain PM165 isolated from pigs in Brasil, and strain XT2008 isolated in China. The results of our study present the molecular characterization of PrV, PPV and PCV2 identified in swine in Republic of Montenegro. Besides that, these results confirmed that PCR is a very useful method for rapid detection of these viruses in subclinically infected swine.

Examination of the influence of different adjuvants on the immunogenicity of vaccines against parainfluenza 3 virus in cattle

Cilj naših istraživanja je bilo ispitivanje uticaja različitih adjuvanasa na imunogenost vakcine protiv parainfluence virusa 3 kod goveda. U ispitivanjima je korišćena komercijalna vakcina Respi-ol protiv virusa PI3 goveda, proizvođača VZS iz Subotice i eksperimentalna vakcina protiv navedenog virusa pripremljena sa gotovim adjuvansom Emulsigen-om. Ispitivanja su vršena na dve ogledne grupe teladi od po 12 životinja koje su dvokratno imunizovane u razmaku od 21 dan. Treća grupa teladi od ukupno 6 životinja je služila kao kontrola u ispitivanjima. Rezultati izvršenih ispitivanja su pokazali da je manja količina adjuvansa Emulsigen-a po dozi vakcine omogućavala postizanje zadovoljavajuće visine titra virus neutralizujućih antitela u uzorcima krvnog seruma vakcinisanih teladi u poređenju sa propisanom, većom količinom standardnog uljnog adjuvansa sadržanog u komercijalnoj vakcini Respi-ol.

Isolation and Molecular Detection of Bovine Parainfluenza Virus Type 3 in Cattle in Serbia

Abstract The presence of bovine parainfluenza virus type 3 (BPIV3) was examined in 119 nasal swabs collected from cattle with severe respiratory infection. All samples were conducted for virus isolation on the MDBK cell line. The cytopathic effect was observed after 48h to 72h in cells inoculated with eight samples (8/119; 6.7%). The confirmation of isolated strains of BPIV3 was done by the virus-neutralization test. In addition, all samples of bovine nasal swabs were also examined for the presence of BPIV3 virus using RT-PCR with primers specific for the part of HN gene. The presence of BPIV3 was detected in eight samples (8/119; 6.7%) that were also positive upon virus isolation. The molecular characterization based on nucleotide sequencing of the part of the HN gene showed that all BPIV3 isolates belonged to genotype C of BPIV3. They branched in one distinct cluster with three different branches, but these branches were very similar to each other (98.1% to 99.8%). Serbian BPIV3c isolates were most similar to the Chinese BPIV3c isolates SD0805, SD0809 and SD0835 (from 97.92% to 99.7%), and to South Korean (12Q061), Japanese (HS9) and American (TVMDL16 and TVMDL20) BPIV3c strains (from 97.1% to 98.8%), and distinct from American (TVMDL15and TVMDL17) and Australian (Q5592) BPI3V genotype B strains (only 79.9% to 82.3% similarity), as well as from the genotype A BPIV3 strains from different countries published in GenBank.

Examination of some biological properties of glycoprotein subunits of PHY-LMV.42 strain of Newcastle disease virus

The objective of our work was to investigate some biological characteristics of purified glycoprotein subunits of Newcastle disease virus strain PHY-LMV.42 isolated from pigeons for the purpose of vaccine production. PHY-LMV.42 strain of Newcastle disease virus was multiplied by successive passages in embryonated eggs and identified by the methods of Reverse transcriptase PCR and Real-Time PCR along with F gene sequencing. Proving the presence of HN and F antigene in the virus subunits samples was carried out by hemagglutination inhibition method with referent immune sera. Biochemical characterization of glycoprotein subunits was performed by SDS-PAGE method as well as liquid chromatography with mass spectrometry (LC ESI-TOF-MS/MS). Testing for the virus subunits immunogenicity was carried out in biological experiment on 75 laying hens Tetra-SSL and 25 chickens Isa Brown by inducing an artificial infection with Hertz 33 strain of the virus. Low concentrations of the virus antigens of 0.36 mg/ml along with glycoprotein fractions of 77 i 58 kDa manifested a strong hemagglutination activity of 4096 HJ/0,1ml. The subunit vaccines of 256 and 128 HJ/0.5 ml induced a protective immune response in all the vaccinated animals. Based on the obtained results it can be concluded that low concentrations of purified virus subunits of PHY-LMV.42 strain can be used for preparing of effective vaccines. [Projekat Ministarstva nauke Republike Srbije, br. TR 31008: Development and application of molecular methods based on polymerase chain reaction (PCR) in quick and direct identification of Newcastle disease virus strains and investigation of immunogenicity of subunit vaccine prepared of their antigens]

Study of the presence of specific Salmonella Enteritidis antibodies in chicken egg yolks by competitive cELISA method.

One of the most common causes of salmonellosis of man and poultry is Salmonella Enteritidis which is often found in the digestive system of adult birds. The infected birds do not display any evident clinical symptoms and, at the same time, they excrete the bacteria into the surrounding environment. Studies are carried out by standard microbiological procedures which include the isolation of Salmonella spp. in egg yolks and their serologic typization by agglutination on microplates. Along these methods, studies on the possibility to use an enzyme immunoassay, such as cELISA, in order to detect the presence of specific antibodies on Salmonella Enteritidis in egg yolks are carried out intensively. The presence of specific antibodies for Salmonella Enteritidis is detected in egg yolk samples from vaccinated flocks resulted in specific positive for a total of 72.22%. Egg yolk samples originating from hens of an unknown immunologic status were cELISA positive in a total of 1.66%. However, egg yolk samples from non-vaccinated hens were positive on the presence of specific antibodies for Salmonella Enteritidis in 23.07% cases. Bearing in mind that standard bacteriological methods did not confirm the presence of Salmonella Enteritidis in egg yolk samples and that cELISA did establish the presence of specific antibodies in the tested samples it can be concluded that cELISA is a more sensitive test.

Implementation of polymerase chain reaction (PCR) and Real-Time PCR in quick identification of bovine herpesvirus 1.

Examinations were performed on 65 samples of nasal smeas taken from calves and young cows with clinical symptoms of respiratory infection to determine the presence of the bovine herpes virus 1 using parallel implementation of molecular and standard methods of virological diagnostics. The appearance of a cytopathogenic effect (CPE) was not established in inoculated cell lines 24h, 48h and 72h following inoculation, or after two successive passages of the examined material sample through these cell lines. The application of polymerize chain reaction (PCR) using a primer for glucoprotein B and thymidine - kinasis, established the presence of bovine herpes virus 1 nucleic acid in one sample of a bovine nasal smear, while the presence of this virus was established in three samples in an examination of the nasal smear samples using the Real-Time PCR method.