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    Genetic Variability of Bovine Viral Diarrhea Virus in the 5 '-UTR in the Central Anatolia of Turkey
    (Univ Fed Rio Grande Do Sul, 2012) Sarikaya, Baki; Azkur, Ahmet Kursat; Gazyagci, Serkal; Aslan, Muhammet Eren
    Background: The genus Pestivirus in the family Flaviviridae comprises the members bovine viral diarrhea virus type 1 (BVDV-1), classical swine fever virus and border disease virus. The BVDV enveloped and the genome is a single-strand positive sense RNA molecule of approximately 12.3 kilobases in length. The genome is transcribed as a single open reading frame, flanked by 5' and 3' untranslated regions. Genetic typing of BVDV has usually been performed using sequences from the 5'-UTR, N-pro and E2 regions. BVDV is an RNA virus with a high genome variability having practical consequences on epidemiology, diagnosis and disease control. Genetic monitoring was suggested as the first step in BVDV control because genetic typing of BVDV shows evidence of an increasing number of variants. For this reason circulating genetic typing of BVDV is important update these data. Circulating BVDV in the field shows genetic and antigenic diversity. 5'-UTR nucleotide sequence analysis has been widely used for pestivirus genotype identification. To further characterize the BVDV, the nucleotide sequence of the 5'-UTR that represents a conserved region of the virus genome was analyzed in many studies. The purpose of the current study was to investigate genotypes of pestivirus were circulating in cattle populations in Central Anatolia Region of Turkey. Materials, Methods & Results: Blood samples from 160 animals in randomly selected seven cattle dairy farms that lives with more than 1100 cattle, were collected between November 2009 and March 2010 from Kirikkale (n = 57), Corum (n = 50), Ankara (n = 21), Yozgat (n = 17), Kirsehir (n = 15) cities where are located in Central Anatolia region of Turkey. To detect BVDV in cattle, viral RNA was extracted from whole blood samples using QIAamp Viral RNA Kit and the 5'-UTR were targeted using RT-nested PCR accomplished with first round primers pair panpestivirus and with second round BVDV-1a, BVDV-1b and, BVDV-2 pooled blood samples, respectively. It was detected in second round of RT-nested PCR that BVDV-1a and, BVDV-2 rate are 0.625%, 7.5% in the cattle respectively but not BVDV-1b. Positive PCR amplicons were purified from agarose gel by using commercial DNA purification kit GeneClean III. Two panpestivirus positive PCR amplicons were sequenced using 326 primer. To determine genetic typing of circulating BVDV in the cities, two panpestivirus positive PCR amplicons were sequenced to found genetic diversity and all data were deposited in GenBank under accession numbers; BVDV/Turkey/Kirikkale/01 (HQ393488.2) and BVDV/Turkey/Kirikkale/02 (HQ393489.2). Gene sequences were compared to Mega 4.1 and ClustalW analyzing software. Discussion: The BVDV has a world-wide distribution and causes significant economical losses especially on cattle farms. In this study, it was investigated genetic variability of BVDV subtypes by identifying the 5'-UTR nucleotide sequences of two panpestivirus amplicons from field samples. It was found that BVDV-1a and BVDV-2 in terms of BVDV epidemiology is genotyping, 0.625% and 7.5% using RT-nested PCR respectively. Genetic typing is important for the precise classification and molecular epidemiology of BVDV-1 and epidemiological information on currently epidemic viruses is also important for BVDV prevention and control. We suggest that vaccines should contain at least one strain of both species in Turkey. The study of genetic diversity of BVDV is useful for the understanding of pestivirus field locations as well as for epidemiological studies and planning future BVDV control and vaccination programs in Turkey.
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    Inactivated Bovine Viral Diarrhea Virus Vaccine Trigger Leucopenia and Lymphopenia on Calves
    (Univ Fed Rio Grande Do Sul, 2011) Sarikaya, Baki; Azkur, Ahmet Kursat; Gazyagci, Serkal
    Background: Bovine viral diarrhea virus (BVDV) in cattle is a very common viral infection that causes economic losses. In acute infection fever, leucopenia and thrombocytopenia may be observed. BVDV, an enveloped, single-stranded positive RNA virus, is a member of the genus pestiviruses within the family of flaviviridae. Vaccination and eradication programs should be applied against BVDV in herds with high prevalence of BVDV that includes removal of persistently infected (PI) animals from the herds. The vaccines used against BVDV are either modified live virus (MLV) or inactivated-virus vaccines. These commercially produced vaccines are being tested before introduced to the market, although afterwards some have been withdrawn regardless of preliminary tests. For example in Germany in 2010, inactive vaccines were withdrawn from the market when 80% of the newborn calves from vaccinated cattle were hemophilia. This phenomenon indicates the side effects of vaccine were needed by independent laboratories. For these reason in this study, in a dairy farm in 23 calves were investigated the effect of vaccination on the blood values. Materials, Methods & Results: In this study it were used 23 healthy heifers aged 6-12-months old, held in a dairy farm in Kirikkale. All of the heifers were vaccinated subcutaneously with one vial of commercial PregSure BVD inactive vaccine as recommended by the manufacturer. Whole blood samples collected before one week and three weeks after one dose commercial inactivated BVDV vaccination, blood values analyzed and compared. Before and after one week from vaccination, the blood values of hematocrit, hemoglobin, leucocytes, red blood cell, lymphocyte, neutrofil/granulocyte and mean corpuscular hemoglobin concentration were decreased and this decrease was statistically significant (P <= 0.05). Before and after three weeks from vaccination mean corpuscular hemoglobin concentration increase was significant (P <= 0.05). One and three weeks after vaccination were compared, hematocrit, hemoglobin and red blood cell values were decreased and white blood cell, lymphocyte and neutrofil/granulocyte values increased found significant (P <= 0.05). Divided into three groups against to BVDV antigen and antibodies in the serum of samples could not be found. Discussion: In the present study we compared to effect of inactivated BVDV vaccination on blood values analyzed and compared with kinetics. Before study it was confirmed that all animal did not have BDVD specific antibodies by Porquier ELISA. When Before and after one week from vaccination, the blood values of hematocrit, leucocytes, lymphocyte, and mean corpuscular hemoglobin concentration were decreased and this decrease was statistically significant (P <= 0.05). According to these results we found that a single-dose of vaccination causes a partial leucopenia and lymphopenia. To investigate whether vaccinations suppress immune system in calves, number of Treg cell population might be more detail observed after vaccination. As a result, though one dose of inactive BVDV vaccine cause lymphopenia and leucopenia it was unable to achieve high titers of antibodies. However veterinarian and animal owner prefer to perform widespread usage of one dose inactive vaccination in Turkey in order to cheaper than multiple dose vaccination.