Phylogenetic analysis of peste des petits ruminants virus from outbreaks in Turkey during 2008–2012

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats and is endemic in all regions of Turkey. In this study, PPR virus infection was investigated by RT-PCR assay based on the fusion (F) gene in PPR-suspected sheep and goat samples. PPR virus RNA was detected in 65 small ruminants (51 sheep, 14 goats) from independent outbreaks during 2008-2012 in provinces in the central and Mediterranean regions and the central-west part of the Aegean region in Turkey. The virus was detected in an aborted sheep fetus sample by RT-PCR, and diagnosis was also confirmed by virus isolation. Vaccine strain Nigeria 75/1 was differentiated from field isolates by restriction fragment length polymorphism analysis of RT-PCR products using EcoRI. Phylogenetic analysis of 16 viruses indicated that all viruses, including the one from the aborted sheep fetus, belonged to lineage IV, as had the PPR viruses previously isolated in Turkey. Nucleotide sequence identity among 16 viruses was 99.1%-100%. Results showed that PPR virus lineage IV has been in circulation in Turkey since the first detection of the disease

Phylogenetic analysis of peste des petits ruminants virus from outbreaks in Turkey during 2008-2012

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats and is endemic in all regions of Turkey. In this study, PPR virus infection was investigated by RT-PCR assay based on the fusion (F) gene in PPR-suspected sheep and goat samples. PPR virus RNA was detected in 65 small ruminants (51 sheep, 14 goats) from independent outbreaks during 2008-2012 in provinces in the central and Mediterranean regions and the central-west part of the Aegean region in Turkey. The virus was detected in an aborted sheep fetus sample by RT-PCR, and diagnosis was also confirmed by virus isolation. Vaccine strain Nigeria 75/1 was differentiated from field isolates by restriction fragment length polymorphism analysis of RT-PCR products using EcoRI. Phylogenetic analysis of 16 viruses indicated that all viruses, including the one from the aborted sheep fetus, belonged to lineage IV, as had the PPR viruses previously isolated in Turkey. Nucleotide sequence identity among 16 viruses was 99.1%-100%. Results showed that PPR virus lineage IV has been in circulation in Turkey since the first detection of the disease

Antigenic and genomic relatedness of turkey-origin coronaviruses, bovine coronaviruses, and infectious bronchitis virus of chickens

In earlier studies in our laboratory, we found that bovine coronavirus (BCV) was pathogenic for 1-day-old turkey poults. This finding prompted us to study the antigenic and genomic relatedness of turkey, origin coronaviruses (TOCVs) to BCV A one-step reverse transcription (RT -polymerase chain reaction (PCR) targeting a 730-base pair fragment of the nucleocapsid (N) gene of BCV and a nested PCR targeting a 407-base pair fragment of the N gene were used in an attempt to detect TOCV from North Carolina, Indiana, and a prototype turkey coronavirus (TCV) obtained from the American Type Culture Collection, Both the one-step RT-PCR and the nested PCR amplified cell culture-passaged isolates of calf diarrhea strains of BCV but none of the 15 tested TOCVs or transmissible gastroenteritis coronavirus of swine. TOCVs also did not cross-react in a BCV antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA) system with monoclonal antibodies (MAbs) against N, spike glycoprotein, and hemagglutinin esterase glycoprotein proteins of BCV as coating antibodies. The same TOCVs could be detected with primers designed from the genome of infectious bronchitis virus (IBV of chickens. These primers amplified it 1082-base pair region spanning portions of thc membrane glycoprotein (M) and N protein genes of IBV and TCV. The TOCVs also cross-reacted in an AC-ELISA with MAbs against the M and subunit 2 of spike glycoprotein of IBV

Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in northwestern Turkey

Bovine coronavirus (BCoV) is an important cause of diarrhoea in calves, winter dysentery in adult cattle and respiratory tract disease in feedlot cattle. Serum, faecal and nasal swab samples were collected from a total of 96 cattle with clinical signs in 29 barns of 23 villages in Northwestern Turkey. The cattle were subdivided into 3 distinct age groups (0-30 days old, 4-12 months old and 2-7 years old). An indirect antigen-capture ELISA and an antibody-detection ELISA as well as geometric mean BCoV antibody titres were used to detect BoCV shed in the faeces and in the nasal secretions, respectively. Relationships between BCoV shedding and age group, seroconversion and clinical signs in cattle were also analysed. The rate of faecal shedding of BoCV was 37.1% (13/35) in 0-30 days old calves, 25.6% (10/39) in 4-12 months old feedlot cattle and 18.2% (4/22) in 2-7 years old cows. The overall rate of BCoV faecal shedding was 28.1% (27/96) in the cattle examined. Only one animal in the 4-12 months old age group was found to shed BoCV nasally. The analysis showed that there was a significant difference (P < 0.0001) with respect to faecal shedding between the clinical signs and the age groups. BCoV antibody titre in 50% of all cattle was <= 100 as detected by ELISA while 27.1% of the cattle had high titres ranging between 1,600 and 25,600. The seroconversion rate was 7.3% (7/96) in animals shedding BoCV in the faeces and 42.7% (41/96) in cattle negative for faecal shedding as detected by ELISA, and 20.8% of cattle with no seroconversion shed BCoV in the faeces. There was no statistically significant association between seroconversion and nasal or faecal BCoV shedding. These findings confirm the presence of BCoV infections in Turkey. Further studies are needed to isolate BCoV strains in Turkey and to investigate their antigenic and genetic properties

Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates

It is unclear whether respiratory and enteric bovine coronavirus (BoCV) strains are distinctive in biological, antigenic and genetic characteristics. In the present study, we analyzed the nucleotide and amino acid sequence of the S I subunit of the S glycoprotein, including the cleavage site, of both respiratory (n = 5) and enteric (n = 3) BoCV isolates including two paired isolates from the same feedlot animals and compared them with the prototype Mebus and two enteric and one respiratory BoCV strains from Quebec. A total of 75 polymorphic nucleotides were identified in the Sl subunit of the spike glycoprotein of BoCV isolates compared with the Mebus strain. These polymorphisms led to 42 amino acid changes at 38 distinct sites. The amino acid changes were distributed throughout the Sl subunit with clustering around residues 40-118, 146-179, and 458-531. Among these variations, only 19 amino acid substitutions altered the charge, hydrophobicity and surface probability of the protein. Based on phylogenetic analysis, our respiratory and enteric isolates clustered into two major groups with two subgroups. Although, there were only a few amino acid changes between the respiratory and enteric paired isolates, the other two respiratory isolates, one isolated from the same farm as a paired strain and the other from a different farm, showed more sequence diversity, Amino acid alterations in residues 113, 115, 118, 146, 148, 501, 510 and 531 of respiratory isolates conferred significant changes in the predicted secondary structure compared with the prototype winter dysentery (WD) and the calf diarrhea (CD) strains of BoCV. In conclusion, the data suggests that respiratory strains of BoCV may differ genetically from the classical calf enteric and adult WD strains. (C) 2002 Elsevier Science B.V. All rights reserved

Methicillin and aminoglycoside resistance in Staphylococcus aureus isolates from bovine mastitis and sequence analysis of their mecA genes

Although methicillin-resistant Staphylococcus aureus (MRSA) were generally isolated from human beings; these agents were recently isolated from various animal species. It has been shown that MRSA isolates are not only resistant to beta-lactam antibiotics, but can also be resistant to the other commonly used antibiotics. In this study, 18 phenotypic methicillin resistant S. aureus isolates from bovine mastitis cases were analyzed by PCR for the presence of mecA gene encoding methicillin resistance and aac(6')/aph(2aEuro(3)), aph(3')-IIIa and ant(4')-Ia genes encoding aminoglycoside resistance. Out of 18 S. aureus isolates (oxacillin MICs, a parts per thousand yen4 mu g/ml), 3 were positive for mecA gene. Only one from 3 mecA positive isolates was positive for genes encoding aminoglycoside-modifying enzymes and this isolate carried aac(6')/aph(2aEuro(3)) in combination with aph(3')-IIIa gene. The aph(3')-IIIa gene was detected in 3 isolates. These three isolates carrying the aminoglycoside-modifying enzyme genes were resistant to gentamicin, kanamycin and neomycin. The mecA gene of 3 MRSA isolates was sequenced. All three mecA genes of these isolates were identical to that found in human MRSA strains, except a one-base substitution at nucleotide position 757. From the data presented in this study, it can be concluded that MRSA isolated from bovine mastitis may be originated from human beings, but further studies are needed to investigate the possibility of zoonotic transfer of MRSA

Molecular and serological investigations of the Influenza A(H1N1) 2009 pandemic virus in Turkey

Intense research has been conducted on influenza A(H1N1)pdm09 virus to determine the virulence markers. Limited information on characteristics of pandemic virus has become available in Turkey since the pandemic. In this first report from Turkey, we investigated the molecular markers that have been associated with increased virulence and oseltamivir resistance. We also conducted serological studies in people after infection, vaccination, exposure, and no-exposure controls to determine the level of protection against the pandemic H1N1 influenza virus. Thirteen rRT-PCR positive samples were analyzed for presence of mutations that have been associated with host range, virulence, and antiviral resistance: substitution D222G in the HA, E627K in the PB2, and H275Y in the neuraminidase (NA). In addition, 135 serum samples from vaccinated, recovered, asymptomatic contacts, and control individuals were tested using hemagglutination inhibition (HI) assay. D222G was detected in nasal samples from two severe cases. No specified mutations in the PB2 and NA were identified. Additional substitutions, I216V, V321I, E374K, S203T in HA, V655I in PB2, and I163V in NA, were detected. HI testing from vaccinated individuals, recovered patients, asymptomatic contacts, and control individuals showed that 97.9, 99.7, 88.2, and 44.2 % had HI titers a parts per thousand yen40, respectively. Molecular markers promoting influenza A(H1N1)pdm09 to become a pandemic virus are still under investigation. Serological results confirm that younger, un-exposed individuals are at increased risk of pandemic virus infections. Influenza A(H1N1)pdm09 viruses are still in circulation around the globe. Therefore, these viruses need to be monitored closely for development of new markers including antiviral resistance mutations

Effects of dietary mannanoligosaccharide on performance, some blood parameters, IgG levels and antibody response of lambs to parenterally administered E-coli O157 : H7

Forty- eight male lambs were used to evaluate the effect of dietary supplementation of mannanoligosaccharide ( MOS) with or without parenteral Escherichia coli injection on their growth performance, feed conversion efficiency, blood metabolites, total serum immunoglobulin G ( IgG) levels and antibody response. Lambs were randomly assigned to four groups of 12 animals each. In groups C ( control) and CE ( E. coli challenged), animals were fed commercial concentrate pellets and hay ( 50: 50), and in groups M ( MOS) and ME ( MOS + E. coli challenged), animals were fed commercial concentrate pellets including MOS at 0.2% and hay ( 50: 50). At day 15 and 30, animals in groups CE and ME were injected subcutaneously with 1 ml of phosphate buffered saline ( PBS) suspension containing 10 6 cfu of heat inactivated non- toxigenic E. coli O157: H7, while animals in C and M groups were injected subcutaneously with 1 ml of PBS. The experimental period was 45 days. Data indicated that body weight of lambs at the end of the study were statistically non- significant among the groups. Blood metabolites, i. e. total protein, albumin, calcium and phosphorus concentrations were not affected significantly by MOS supplementation. However, administration E. coli lowered ( p < 0.05) total protein, albumin and calcium concentrations in the serum on day 30. The IgG level was not different between groups. However, on day 45, the total IgG level was found to be higher ( p < 0.05) in lambs that had received MOS and E. coli than in other groups. Application of MOS did not have any effect on the antibody response to E. coli as OD values

Circulation of bovine ephemeral fever in the Middle East-Strong evidence for transmission by winds and animal transport

Bovine ephemeral fever virus (BEFV) is an economically important arbovirus of cattle. The main routes of its transmission between countries and continents are not completely elucidated. This study aimed to explore BEFV transmission in the Middle-East. A phylogenetic analysis was performed on the gene encoding the G protein of BEFV isolates from Israel from 2000 and 2008 with isolates from Turkey (2008), Egypt (2005), Australia (1968-1998) and East Asia (1966-2004). Calf sera collected during the years 2006-2007 were tested by serum neutralization in order to explore for recent exposure to BEFV before 2008. These were followed by a meteorological analysis, aimed to reveal movement of air parcels into Israel in the two weeks preceding the first case of BEF in Israel in 2008. The 2008 Israeli and Turkish isolates showed 99% identity and formed a new cluster with the 2000 Israeli isolate. The serological survey showed no new exposure to BEFV during 2006 and 2007. These results coincided with the meteorological analysis, which revealed that air parcels originating in Southern Turkey had reached the location of outbreak onset in Israel nine days before the discovery of the index case. The Egyptian isolate clustered phylogenetically with the Taiwanese isolates, coinciding with data on importation of cattle from China to the Middle East in the year preceding the isolation of the Egyptian isolates. These results suggest that both winds and animal transport may have an important role in trans-boundary transmission of BEFV. (C) 2012 Elsevier B.V. All rights reserved

Surveillance and Oseltamivir Resistance of Human Influenza A Virus in Turkey During the 2007-2008 Season

Monitoring the activity of influenza viruses is important for establishing the circulating types and for detection of the emergence of novel sub-types and antiviral resistant strains. This is the first report from Turkey on the surveillance and oseltamivir resistance of influenza viruses in 2007-2008. Five hundred twenty-four nasal swabs were tested from different geographical regions in Turkey during November 2007-April 2008. One hundred sixty-three (31%) samples were positive for influenza viruses of which 111 (68%) were influenza A, 52 (31%) influenza B using an immuno-capture ELISA. Forty isolates were selected at random from influenza A positive samples and grown in MDCK cell cultures. The supernatant of the cell cultures was used for RNA extraction followed by RT-PCR to detect the sub-types. Sub-typing revealed all samples as A/H1N1. The N1 gene segment of 30 A/H1N1 samples was sequenced in part, from the 201st to 365th residue, which included the critical region for oseltamivir resistance. Then resulting sequences were analyzed with oseltamivir sensitive and resistant strains obtained from National Center for Biotechnology Information (NCBI) GenBank by CLC Main Workbench Software. H275Y (H274Y according to N2 numbering) mutation, which is known to confer resistance to oseltamivir, was detected in 6 out of 30 (20%) H1N1 isolates from four cities (Istanbul, Bursa, Ankara, and Izmir). The D354G mutation was observed in all oseltamivir resistant H1N1 isolates but not in the oseltamivir sensitive isolates. Assay of neuraminidase activity revealed that these isolates were resistant to oseltamivir, but sensitive to zanamivir. J. Med Virol. 81:1645-1651, 2009. (C) 2009 Wiley-Liss, Inc