In the pilot study, after the detection of virus-positive milk tanker samples, one lactating cow was identified as PI cattle on Farm No. milk. In the regional survey, Dihydrostreptomycin sulfate 5 of 79 milk tanker samples were virus-positive. The virus was detected in three PI lactating cows and one PI calf on three farms. Antibody screening using bulk tank milk samples revealed 15 of 363 samples were positive, and 12 of 348 farms were BVDV antibody-positive. Follow-up tests on one farm identified three PI calves. Thus, eight PI cattle on five farms were identified in this study. In conclusion, combining BVDV detection using milk tanker samples and antibody detection using bulk tank milk is a feasible and economical method to efficiently screen PI cattle and confirm the PI-free status among dairy herds. Keywords:antibody enzyme-linked immunosorbent assay, bovine viral diarrhea virus, milk tanker, persistently infected cattle, reverse-transcription polymerase chain reaction Bovine viral diarrhea (BVD) is a chronic infectious disease in cattle caused by infection with the BVD virus (BVDV) [10]. It results Dihydrostreptomycin sulfate Dihydrostreptomycin sulfate in significant economic losses to the cattle industry worldwide [9,14,33]. Clinical signs are frequently fever approximately 69 days postinfection, inappetence, and mucosal lesions; however, most cases of BVDV infection are subclinical in susceptible nonpregnant cows [23]. If clinical signs are severe, acute BVD infection may be followed by reproductive disorders immediately or shortly after seroconversion because of the general condition of the cow [15]. Moreover, transplacental infection can cause fetal death and abortion, significant fetal abnormalities [6,7], or the birth of persistently infected (PI) calves [3,30]. BVDV can be transmitted to the fetus in pregnant cattle during days 40120 of gestation, and thereby induces fetal immune tolerance to BVDV and results in the delivery of PI calves [3,5]. The PI calves continuously excrete large amounts of virus throughout their lives, show few symptoms, and are a continual source of infection in a herd [13,18,24]. Furthermore, they are at risk of developing fatal mucosal disease [4,5]. Infection with BVDV has severely damaged the cattle industry throughout Japan. Based on the results of the latest survey on PI animals on dairy farms in Japan, the prevalence of farms with PI animals is calculated as 7.6% (95% confidence interval [CI], 3.116.4%), and the prevalence of cattle tested as PI animals was 0.12% (95% CI, 0.050.25%) [17]. In addition, based on the investigation of BVDV epidemics from 2006 to 2014 in Hokkaido, where most dairy cattle are reared in Japan, BVDV-1b and BVDV-2a viruses were the predominant BVDV subgenotypes [1]. The reverse-transcription polymerase chain reaction (RT-PCR) technique has been used to detect BVDV-infected cattle among dairy herds. This testing technique for bulk tank milk is useful as a screening test to detect any PI cattle among lactating cows [19]. It is a cost-effective diagnostic method. The virus has been detected by RT-PCR when milk from a PI animal was diluted Dihydrostreptomycin sulfate to 1 1:600 with milk from a BVDV-free herd [28]. In Nordic countries such as Denmark, Sweden, Norway, and Finland where BVDV vaccines are not used in BVDV control programs, antibody detection in bulk tank milk by using indirect or blocking enzyme-linked immunosorbent assays (ELISAs) has been used as a diagnostic tool for monitoring the BVDV infection-free status [15,25]. Houeet al.[15] revealed that PI animals were found only among young stock, and that PCR analysis of bulk tank milk was therefore unsuitable to test a herd for the presence of PI animals. Furthermore, they stated that, because of the high sensitivity and low specificity of bulk tank milk antibody testing, this method identifies nearly all true-positive herds, but tends to produce a certain number of false-positive herds [15]. Thus, for a BVDV control program, combining several available diagnostic tests that can detect the virus itself or detect viral-specific antibodies is important to improve the accuracy of detecting PI cattle. In consideration of the more effective and rapid assessment of BVDV status in dairy herds, we focused on screening for BVDV in milk tanker samples. The milk tanker samples, which include commingled milk collected from several dairy farms, are often used to detect antibiotics to prevent them from getting mixed into the plant [31]. For pathogen screening, milk tanker samples are used to detectListeriaspp. [29]. Milk tanker samples save the labor involved in sampling bulk tank milk or serums on each dairy farm. Therefore, the milk tanker samples have the potential to screen BVDV from cattle on several dairy farms simultaneously. However, whether milk tanker samples Dihydrostreptomycin sulfate are sufficiently sensitive to identify PI animals among dairy herds has not been fully studied. Our objective was to provide a screening scheme of PI Tek cattle in dairy herds by combining RT-PCR to detect BVDV in milk.