sample collection : samples were mainly collected in kirikkale [ ( 3950 n ; 
 3331 e ; altitude 700 m ) ] . there were 45,664 dairy cattle in kirikkale and approximately 
 13,000 cattle over 15 months of age . a low rate of abortions is usually observed on farms , 
 and 3 abortions per 100 pregnancies per year are often considered a  normal the blood samples were collected from 390 dairy cattle over 15 months of age with a history 
 of abortion and/or infertility from randomly selected herds in kirikkale province . the remaining blood samples were collected in ankara ( 3956 n ; 3252 e ; altitude 870 m ; 
 n=91 ) , corum ( 3914 n ; 3827 e ; altitude 810 m ; n=86 ) and yozgat ( 3950 n ; 3448 e ; 
 altitude 1418 m ; n=89 ) provinces , which all border kirikkale province . whole blood samples were collected into edta - coated tubes and stored in 20c until rna or 
 dna isolation . blood samples were collected into tubes without an anticoagulant for 
 separation of serum , allowed to clot at room temperature and centrifuged at 2,000 rpm for 10 
 min . serological tests for detection of brucellosis : rbpt and stat were 
 performed according to the procedures described by the manufacturer of the kits ( pendik 
 institute , istanbul , turkey ) . the stat was considered positive , if there were any signs of 
 agglutination in the rbpt and the titer was1:80 . elisa : commercial elisa kits were used according to the manufacturers 
 instructions to detect the antibodies against bvdv ( bvdv ab elisa , idexx , westbrook , me , 
 u.s.a . ) , bhv-1 ( herdchek ibr ge , idexx ) and bhv-4 ( bio - x diagnostics , rochefort , belgium ) 
 and the antigen of bvdv ( elisa bvd / md antigen mix screening , institut pourquier , 
 montpellier , france ) . optical densities at appropriate wavelengths were measured by an 
 automated elisa reader ( sirio s elisa reader , reiged diagnostics , jakarta , 
 indonesia ) . pcr and rt - pcr : to detect the bhv-1 , bhv-4 or bhv-5 gene , dna was 
 extracted from blood using a spin column system ( high pure pcr template preparation , roche , 
 mannheim , germany ) according to the manufacturer s instructions . to detect the bhv-1 gb , bhv-4 gb and bhv-5 gd genes , pcr was performed with the primers 
 shown in table 1table 1.primers used for rt - pcr and pcr in this studyvirusprimer name5 3length of base pairreferencepan - pestivirus324atgcccwtagtaggactagca288 bp326tcaactccatgtgccatgtacbvdv-1a1aftcgacgccttrrcatgaaggt169 bpbvdv-1b1bftcgacgctttggaggacaagcbvdv- 1a/1b1abrccatgtgccatgtacagbvdv-2b5actagcggtagcagtgag221 bpb6ctagcggaatagcaggtcbhv-1bhv-1gb1aagcgcaaaaacgtgtg323 bpbhv-1gb2tgcaggtacagcttggcbhv-4bhv-4gb1cccttctttaccaccacctaca615 bpbhv-4gb2tgccatagcagagaaacaatgabhv-5bhv-5gd1gcccgcagtttcccctacc564 bpbhv-5gd2cgcacccgctctcaatctt . the mixture for all pcr reactions contained 5 l dna , 25 mm 
 tris - hcl ( ph 8.9 ) , 3 mm mgcl2 ( mbi fermentas , vilnius , lithuania ) , 2 mm of dntp 
 mix ( mbi fermentas ) , 10 pmol of each primer and 1 u of taq 
 dna polymerase ( mbi fermentas ) . the reaction conditions for pcr were as follows : 95c for 2 
 min ; 35 cycles of 95c for 30 sec , 58c , 56c or 59c for 30 sec ( bhv-1 gb ) , 45 sec ( bhv-4 
 gb ) or 1 min ( bhv-5 gd ) , respectively , and 72c for 30 sec ; and finally 72c for 10 min . to detect bvdv gene , rna was extracted from blood using a spin column system ( high pure 
 viral rna kit , roche ) according to the manufacturer s instructions . cdna synthesis and 
 rt - pcr were performed according to sarikaya et al .   with the primers shown in table 
 1 . all pcr or rt - pcr reactions were carried out in a bo - pcr-5 thermal cycler ( boeco , hamburg , 
 germany ) . an ingenius lhr ( syngene , cambridge , u.k . ) was used to visualize pcr amplicons 
 after electrophoresis in 1% agarose gel and ethidium bromide staining . nucleotide sequence analysis and accession numbers : amplified fragments 
 were purified from agarose using a geneclean iii kit ( mp biomedicals , 
 illkirch - graffenstaden , france ) following the manufacturer s instructions . direct sequencing 
 of the dna fragments was performed for each pcr product by a commercial company ( iontek , 
 istanbul , turkey ) . nucleotide sequences were compared with analysis software ( mega 6 and 
 clustal omega ) . the nucleotide sequences were deposited in the genbank database under the 
 accession numbers for bvdv ( fig . whole blood and serum samples were collected from a total of 656 cows in ankara ( n=91 ) , 
 corum ( n=86 ) , kirikkale ( n=390 ) and yozgat ( n=89 ) provinces . forty - five of 656 serum samples 
 ( 6.86% ) were found to be positive for brucella spp . by rbpt , and all 
 rbpt - positive serum samples were confirmed by stat to determine their titers . the titers of 
 41 of the 656 serum samples ( 6.25% ) were found to be1/80 . a total of 615 serum samples 
 ( excluding sera positive for brucella spp . ) were further tested with elisa 
 to determine the seropositivity rate against bvdv , bhv-1 and bhv-4 . the elisa seropositivity rate for bvdv was 70.89% ( 436/615 ) ( table 2table 2.seroprevalence rates for bvdv , bhv-1 and bhv-4 in serum samples negative for 
 brucella spp . the values represent the numbers of seropositive 
 samples detected by rt - pcr and the numbers of pcr - positive samplesvirusprevalence ( % ) antibodiesgenebvdv436/615 ( 70.89%)18/506 ( 3.55%)bhv-1254/615 ( 41.3%)1/254 ( 0.39%)bhv-4177/615 ( 28.78%)0/177bhv-5not performed0/254**pcr performed with 254 bhv-1 seropositive samples for bhv-5 ) . there was limited information about vaccination against bhv-1 in the cattle 
 herds accordingly , all animals were considered vaccinated , 
 and bhv-1 ge elisa was used to distinguish naturally infected cattle from vaccinated cattle . the bhv-1 seropositivity rate was 41.3% ( 254 out of 615 ) ( table 2 ) . the seropositivity rate detected for bhv-4 was 28.78% 
 ( 177 out of 615 ) ( table 2 ) . * pcr performed with 254 bhv-1 seropositive samples for bhv-5 for genotypic determination of bvdv , a total of 506 whole blood samples ( 436 antibody 
 positive and 70 antigen positive by elisa ) were subjected to rt - pcr , and 18 ( 3.55% ) of them 
 were positive ( table 2 ) . when a second round of 
 pcr was carried out with internal primers for determination of subtypes bvdv 1a , bvdv 1b and 
 bvdv-2 , no amplicons were observed . for genome detection of bhv-1 , pcr was carried out in 
 254 seropositive samples , and 1 ( 0.39% ) whole blood sample was found to be positive ( table 2 ) . pcr was performed to test 177 bhv-4 
 seropositive samples for bhv-4 and 254 bhv-1 seropositive samples for bhv-5 , and no 
 amplicons were observed for bhv-4 and bhv-5 ( table 
 2 ) . the prevalences were calculated depending on the provinces in which the serum samples were 
 collected . the seropositivity rates in kirikkale were 316/365 ( 85.47% ) for bvdv , 189/365 
 ( 51.78% ) for bhv-1 and 107/365 ( 29.31% ) for bhv-4 . the seropositivity rates in yozgat were 
 97.46% ( 77/79 ) , 79.74% ( 63/79 ) and 62.02% ( 49/79 ) for bvdv , bhv-1 and bhv-4 , respectively . 
 in ankara , elisa revealed that 43.02% ( 37/86 ) , 2.32% ( 2/86 ) and 24.41% ( 21/86 ) of the 
 samples were positive for bvdv , bhv-1 and bhv-4 , respectively . the seropositivity rate for 
 bvdv in corum was 6/85 ( 7.05% ) , and no specific antibody was detected for bhv-1 and bhv-4 
 ( table 3table 3.seroprevalence of bvdv , bhv-1 and bhv-4 in serum samples negative for 
 brucella spp . according to the provinces sampledprovinceprevalence ( % ) bvdv ab ( + ) bhv-1 ab ( + ) bhv-4 ab ( + ) kirikkale316/365 ( 86.57%)189/365 ( 51.78%)107/365 ( 29.31%)corum6/85 ( 7.05%)0/850/85yozgat77/79 ( 97.46%)63/79 ( 79.74%)49/79 ( 62.02%)ankara37/86 ( 43.02%)2/86 ( 2.32%)21/86 ( 24.41% ) ) . no antibody response against bvdv , bhv-1 or bhv-4 was observed for 143 of 615 ( 23.25% ) 
 samples ( fig . 1.the seropositivity rates of bhv-1 , bhv-4 and bvdv infections in cattle sera . a , 
 single- , double- and triple - positive samples ; b , samples seronegative for bvdv , bhv-1 
 and bhv-4 . ) . however , 111 of 615 ( 18.04% ) 
 samples had antibodies against three of the viruses in combination . the rates of double 
 positivity for bvdv / bhv-1 , bvdv / bhv-4 and bhv-1/bhv-4 were 20.65% ( 127/615 ) , 6.82% ( 42/615 ) 
 and 0.65% ( 4/615 ) , respectively ( fig . the seropositivity rates of bhv-1 , bhv-4 and bvdv infections in cattle sera . a , 
 single- , double- and triple - positive samples ; b , samples seronegative for bvdv , bhv-1 
 and bhv-4 . a phylogenetic tree of bvdv was generated by comparison of the nucleotide sequences between 
 the previously reported 5-utr of the pestivirus genome and our study . the nucleotide 
 sequences found in this study have the highest sequence similarity with hq393488 and the 
 lowest sequence similarity with gu979818 ( fig . the phylogenetic maximum likelihood tree for the 5-utr 
 of the pestivirus genome was generated by using the mega 6 software . sequences 
 generated in this study were as follows : 119-kf425303 , 74-kf434627 , 60-kf434625 , 
 62-kf434626 , 32-kf434623 , 24-kf434622 , 22-kf434621 , 20-kf434620 , 14-kf434618 , 
 16-kf434619 , 12-kf425300 , 45-kf425299 , 413-kf425302 , 414-kf425301 , 90-kf434630 , 
 84-kf434629 , 81-kf434628 and 42-kf434624 . sequences not generated in this study were 
 derived from genbank and were as follows : hq393488 , hq393489 , eu716142 , eu716128 , 
 dq973179 , eu716151 , af298062 , eu716126 , af298069 , af298066 , fj493485 , af299317 , 
 eu716118 , dq973172 and eu716125 for bvdv-1 ; eu542421 and ab042676 for bvdv-2 ; 
 am418428 , fj493487 , gu979820.1 , gu979819.1 and gu979818.1 for bdv ; and aj605590 for 
 csfv . ) . the bhv-1 sequence was deposited in genbank under accession number kf716130.1 . according to 
 phylogenetic analysis , the sequence obtained in this study showed the highest similarity to 
 the reported sequences ef175730.1 and kf584168.1 of the bhv-1 gb gene deposited in genbank 
 ( fig . 3fig . sequences ( ef175730.1 , kj652520.1 , 
 kj652518.1 , kf584168.1 , kc479146.1 , kf601566.1 , kf601565.1 , kc479149.1 , kc479143.1 , 
 jf920419.1 , dq006854.1 , kf734616.1 , kc682098.1 , jn106444.1 , ay330349.1 , eu272475.1 , 
 jf920420.1 , jf920418.1 , eu523744.1 , af258347.1 , dq006857.1 , ef624475.1 , kj652519.1 and 
 jx898220.1 ) were not generated by this study , but were derived from genbank for the 
 kf716130.1 sequence . ) . the phylogenetic maximum likelihood tree for the 5-utr 
 of the pestivirus genome was generated by using the mega 6 software . sequences 
 generated in this study were as follows : 119-kf425303 , 74-kf434627 , 60-kf434625 , 
 62-kf434626 , 32-kf434623 , 24-kf434622 , 22-kf434621 , 20-kf434620 , 14-kf434618 , 
 16-kf434619 , 12-kf425300 , 45-kf425299 , 413-kf425302 , 414-kf425301 , 90-kf434630 , 
 84-kf434629 , 81-kf434628 and 42-kf434624 . sequences not generated in this study were 
 derived from genbank and were as follows : hq393488 , hq393489 , eu716142 , eu716128 , 
 dq973179 , eu716151 , af298062 , eu716126 , af298069 , af298066 , fj493485 , af299317 , 
 eu716118 , dq973172 and eu716125 for bvdv-1 ; eu542421 and ab042676 for bvdv-2 ; 
 am418428 , fj493487 , gu979820.1 , gu979819.1 and gu979818.1 for bdv ; and aj605590 for 
 csfv . the phylogenetic tree for the gb gene of bhv-1 . sequences ( ef175730.1 , kj652520.1 , 
 kj652518.1 , kf584168.1 , kc479146.1 , kf601566.1 , kf601565.1 , kc479149.1 , kc479143.1 , 
 jf920419.1 , dq006854.1 , kf734616.1 , kc682098.1 , jn106444.1 , ay330349.1 , eu272475.1 , 
 jf920420.1 , jf920418.1 , eu523744.1 , af258347.1 , dq006857.1 , ef624475.1 , kj652519.1 and 
 jx898220.1 ) were not generated by this study , but were derived from genbank for the 
 kf716130.1 sequence . the livestock industry is severely affected by production losses due to abortion , neonatal 
 deaths and infertility , which can be prevented by investigations of the causing infections . this study shows that bvdv , bhv-1 , bhv-4 and bhv-5 may play an important role in genital 
 disorders and abortions in cattle in ankara , corum , kirikkale and yozgat provinces , 
 turkey . although noninfectious factors , such as genetic , hormonal and metabolic disorders , feeding 
 problems and unsuitable vaccination programs , cause reproductive problems , most of the 
 reported main factors for abortion , neonatal death and infertility are still due to viral , 
 bacterial , protozoal and fungal infections . the 
 abortions and stillbirths of 8,995 cattle were investigated over a 10-year period , and 2,723 
 ( 30.38% ) of the abortions were determined to have resulted from infectious agents , while 219 
 ( 2.44% ) cases were the result of noninfectious causes . the rates of abortion caused by 
 infection with viruses , bacteria and fungi were 10.57% , 14.49% and 5.31% , respectively . the 
 etiology of the remaining 6,020 ( 67.17% ) abortions could not be determined . at the end of 2011 , schmallenberg virus was 
 announced as a prevalent novel virus that causes abortions in european countries as well as 
 turkey [ 6 , 23 ] . another recently discovered virus , hobi - like virus , was also found to be a reason for cattle 
 abortions and neonatal deaths . these recently 
 discovered viruses could have played a role in undetermined abortions reported in previous 
 studies . in this study , we aimed to focus on viral agents , as there was limited information 
 regarding the sampling area and cause of genital disorders ( abortion , infertility , etc . ) . samples seropositive for brucella were excluded , and a total of 615 
 negative serum samples were tested with commercial elisa kits to detect specific antibodies 
 for bvdv , bhv-1 and bhv-4 . in our study , it was determined that the rate of bvdv 
 seropositivity was 70.89% ( 436 out of 615 ) by elisa , and 3.55% ( 18 out of 506 ) of whole 
 blood samples were positive for the bvdv gene . the seropositivity and antigen ( or genome ) 
 rates for bvdv were 46.2286% and 2.34.9% in previous studies of cattle in turkey , 
 respectively [ 3 , 46 , 58 ] . bvdv-1 and bvdv-2 have been reported in many countries around the world , and bvdv-1a , 1b , 
 1d , 1f , 1h , 1 g and 2a genotypes have already been reported in turkey [ 27 , 37 , 39 , 41 ] . investigated the genetic 
 variability in the 5-utr of bvdv from samples obtained from kirikkale and detected bvdv-1a 
 ( 0.625% ) and bvdv-2 ( 7.5% ) . in this study , bvdv subtypes ( bvdv 1a , bvdv 1b and bvdv-2 ) were 
 investigated with internal primers , but no amplicons were detected . the observed difference 
 between this study and that of sarikaya et al .   suggested that the bvdv circulating among the cattle in the study 
 area showed high relatedness according to phylogenetic analyses , although there was a 
 difference in genotype . hence , although there is a high relatedness between the circulating 
 strains , we speculated that there could be other genotypes besides bvdv 1a , bvdv 1b and 
 bvdv-2 in this area . according to phylogenetic analysis , the bvdv sequences determined in 
 this study were close to those of bvdv-1 previous reported in kirikkale , turkey . the ge blocking elisa is suitable for differentiating between infected cattle and cattle 
 vaccinated with a ge - negative vaccine . comparison of the ge - elisa with the highly sensitive gb - elisa revealed a relative 
 specificity of 99% and a relative sensitivity of 92% . in the present study , previous studies reported a seropositivity rate for bhv-1 of between 
 9.2574% in turkey [ 4 , 5 , 12 , 39 ] . the prevalences of bvdv , bhv-1 and bhv-4 were higher in kirikkale and yozgat in comparison 
 with ankara and corum . the blood samples were collected from dairy cattle herds managed by 
 an intensive cattle production system in ankara and corum , while they were randomly 
 collected in kirikkale and yozgat . the observed results suggested that the intensive cattle 
 production systems may be a limiting factor for the circulation of viral agents amongst 
 dairy cattle farms . analysis of overlapping seroprevalence showed that the rates of double positivity were 
 20.65% for bvdv / bhv-1 , 6.82% for bvdv / bhv-4 and 0.65% for bhv-1/bhv-4 . the seroprevalence 
 rates for bvdv , bhv-1 and bhv-4 in cattle with abortion problems were previously reported as 
 rates of double seropositivity for bhv-1/bvdv ( 23.6% ) and bhv-1/bhv-4 ( 6.4% ) . furthermore , the highest rates of double 
 seropositivity in other studies were 40.2%   and 
 33.33%   for bhv-1/bvdv . thus , it was speculated 
 that the abortion and infertility problems in cattle were mainly caused by bhv-1 and bvdv 
 together . bhv-1 can become latent following a primary infection with a field isolate or 
 vaccination with an attenuated strain in sensory neurons and in non - neural sites [ 11 , 35 ] . stressful 
 conditions , such as transport and parturition and the application of corticosteroids , can be 
 reasons for reactivation of a latent infection . 
 in this study , detection of the bhv-1 gb gene was carried out in 254 samples found to be 
 seropositive for bhv-1 , and only one out of 254 ( 0.39% ) whole blood samples was detected as 
 positive by pcr . bhv-1 circulating in this area was genetically close to bhv-1 isolates 
 reported in india ( ef175730.1 ) and israel ( kf584168.1 ) . this is the first nucleotide 
 sequence of bhv-1 circulating in this area of turkey to be deposited in genbank . calves up to 6 months of age are sensitive for bvh-5 compared with adult cattle , and bhv-5 
 persists in the nervous system or brain tissue after experimental or natural infections 
 . in our study , all anti - bhv-1 
 antibody - positive samples were tested for the bhv-5 gd gene by pcr , but there were no 
 positive results . sampling of older cattle could be one of the reasons for bhv-5 negativity 
 in blood samples . on the other hand , bhv-5 could be present in several nervous system 
 tissues during latent infection . thus , sampling of nervous system tissues could be 
 preferable in future studies to obtain more effective results for detection of bhv-5 
 infection . bhv-4 has been identified in cases of vulvovaginitis , endometritis , mastitis and abortion 
 and also from apparently healthy cattle . several 
 studies reported that the seroprevalence rate for bhv-4 varies between 20.22 and 69.6% in 
 turkey [ 5 , 10 , 
 22 , 28 , 
 57 ] . in our study , the bhv-4 seropositivity rate 
 was 28.78% , and the comparative rates of double seropositivity for bhv-4/bhv-1 and 
 bhv-4/bvdv were 0.51% and 8.2% , respectively . pcr was performed for 177 samples positive for 
 the bhv-4 antibody , but no amplicons were observed for bhv-4 . however , the determined rate 
 of bhv-4 seropositivity may be related to genital system problems in cattle of the study 
 area . in conclusion , bhv-1 , 
 bvdv and bhv-4 , in dairy cattle farms in ankara , corum , kirikkale and yozgat provinces . our 
 observed seropositivity rates could be considered important , as these agents may lead to 
 abortion in cattle in this region . a study of the genetic diversity of bvdv and bhv-1 is 
 useful for understanding the field distribution of pestivirus , for epidemiological studies 
 and for establishment of further preventative measures and vaccination programs in turkey . this study shows the necessity of investigation of these infectious diseases that cause 
 economic losses in dairy farms . therefore , thorough investigation of these infection agents 
 and genotypic characterization of them in the future are crucial to the design of detailed 
 survey studies and development of control measures .
the aim of the study was to determine the epidemiological data of bovine viral diarrhea 
 virus ( bvdv ) , bovine herpesvirus-1 ( bhv-1 ) , bovine herpesvirus-4 ( bhv-4 ) , bovine 
 herpesvirus-5 ( bhv-5 ) and brucella  associated cattle that were previously 
 reported to have abortion and infertility problems in ankara , corum , kirikkale and yozgat 
 provinces , turkey . 
 whole blood and sera samples were obtained from 656 cattle , and 
 antibodies against brucella spp . 
 were detected in 45 ( 6.86% ) and 41 
 ( 6.25% ) animals by rose bengal plate and serum tube agglutination tests , respectively . 
 the 
 seropositivity rates against bvdv , bhv-1 and bhv-4 were 70.89% , 41.3% and 28.78% , 
 respectively . 
 rt - pcr and pcr were performed to detect rna and dna viruses in blood 
 samples , respectively . 
 the bvdv 5-untranslated region and bhv-1 gb gene detected in this 
 study were phylogenetically analyzed . 
 the bvdv strains analyzed in this study were closely 
 related to those previously reported from turkey . 
 the nucleotide sequence from the bhv-1 
 strain detected in this study is the first nucleotide sequence of bhv-1 circulating in 
 this area of turkey deposited in the genbank . 
 the presence of brucella 
 spp . and prevalence of bhv-1 , bhv-4 and bvdv in cattle should be further investigated 
 throughout these regions .