Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves.

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.

Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows.

The objective of our study was to evaluate 2 pregnancy-associated glycoprotein (PAG)-based enzyme-linked immunosorbent assays (ELISAs) for use with either blood or milk. From 12 dairy farms, 116 Montbéliarde or Holstein cows were selected that had either undergone artificial insemination (AI; n = 102) or had calved (n = 14) 2-3 months earlier and had not undergone any further AI. Serum, plasma, and milk were obtained from all cows; serum and plasma were analyzed using the blood pregnancy test and milk using the milk pregnancy test. No false-positive results were observed when samples of the 14 noninseminated cows were tested. Cows undergoing AI were sampled at ~16, 30, and 41 days post-AI. An additional milk sample was taken at ~53 days post-AI. To establish whether the inseminated cows were pregnant, the cows were subjected to transrectal ultrasonography (TU) on or around day 41. Of the 102 inseminated cows, 63 were confirmed pregnant by TU. By day 30, the serum, plasma, and milk ELISAs demonstrated 100%, 100%, and 98.1% sensitivity and 88.6%, 88.9%, and 90.3% specificity, respectively, with potential pregnancy losses 30-41 days post-AI. Accuracy obtained on serum, plasma, and milk at ~41 days post-AI and on milk at ~53 days post-AI ranged from 97.4% to 100%. There were no differences of practical significance in performance between the blood and milk ELISAs for the sampling dates chosen. This new diagnostic capability with milk samples offers a major improvement in bovine reproductive management.

[Pregnancy diagnosis by detection of pregnancy-associated glycopoteins in milk: evaluation of a commercial available ELISA]. / Trächtigkeitsdiagnostik per Milchprobe: Evaluation des Nachweises von trächtig- keitsassoziierten Glykoproteinen mittels kommerziellem ELISA.

In order to increase fertility in modern dairy farming, the interval between first insemination and conception should be as short as possible. Therefore, one approach is to diagnose non pregnant animals early to re-inseminate them as soon as possible. Commercial milk pregnancy assays are available to detect throphoblast derived bovine pregnancy-associated glycoproteins (bPAG) in milk. The aim of the present study was first to evaluate pre-analytical factors interfering with the correct detection of bPAG in milk. To achieve this aim, the stability of the bPAG was tested after repeated freezing and thawing cycles, as well as after a storage of seven days at room temperature and at 37°C. Secondly, the diagnostic performance of a commercially available PAG-ELISA was evaluated for pregnancy diagnosis between 28 to 60 days after artificial insemination by comparing the results with transrectal ultrasonography in a field study (n = 291 cows). After one freezing and thawing procedure the optical density (OD) increased and afterwards stayed stable. The storage of milk samples at room temperature had no effect on the OD, but after five days of storage at 37 °C the OD dropped sharply. It is therefore recommended to add an appropriate microbicidal preservative for shipment and storage of milk samples for bPAG analysis . The results of the field study showed that PAG-ELISA results and ultrasonography results agreed in 95.7% of the cases. Nine samples were tested as "false negative"and five as "false positive", however a re-check revealed that all "false positive"and "false negative" tested cows suffered from embryonic/fetal mortality. In conclusion, the pregnancy diagnostic by bPAG detection in milk samples is an accurate method to diagnose early pregnancy in lactating cows.