Detection of IgM antibodies against bovine viral diarrhea virus using IgM capture ELISA on farms with persistently infected cattle

Abstract

Bovine viral diarrhea (BVD) is a serious disease in cattle and causes economic losses in the livestock industry. Bovine viral diarrhea virus (BVDV) is the causative agent of BVD and spreads among herds via persistently infected (PI) animals that shed large amounts of the virus throughout their lives. Hence, identifying, and culling PI animals and assessing the immune status against BVDV on farms are important strategies for controlling BVD. Additionally, estimating the time when individuals around PI animals were infected with the virus could also be supportive information to interpret a farm status. We herein constructed a BVDV-specific IgM capture ELISA using recombinant E2 antigen and applied it to detecting BVDV-specific IgM antibodies on farms with identified PI cattle. The IgM ELISA detected anti-BVDV IgM antibodies during approximately 2–3 weeks post infection and identified IgM-positive cattle on two farms with recognized PI cattle. Virus neutralization tests showed that almost all adult cattle had high virus neutralization antibodies against BVDV, and sero-positive and -negative cattle coexisted in young herds. In this situation, most of the IgM-positive cattle were in relatively young animals, implying that BVDV had been recently spreading in these young herds. Thus, our findings demonstrated that detecting IgM antibodies could be useful to know recent BVDV infection on farm on which PI cattle were identified.

Highlights

• •We constructed a BVDV-specific IgM capture ELISA using recombinant E2 protein.
• •The constructed assay detected anti-BVDV IgM antibodies during approximately 2–3 weeks post infection.
• •The assay could identify IgM-positive animals on farms with recognized PI cattle, and most of the IgM-positive cattle were in relatively young animals.
• •Our findings demonstrated that detecting IgM antibodies could be useful to know recent BVDV infection on farm with PI animals.

1. Introduction

Bovine viral diarrhea (BVD) is one of the most important infectious diseases in cattle and causes large economic losses in the livestock industry [[1], [2], [3]]. Bovine viral diarrhea virus (BVDV), which belongs to the genus Pestivirus within the family Flaviviridae, is the causative agent of BVD and contains two species of viruses, BVDV1 (Pestivirus bovis) and BVDV2 (Pestivirus tauri). Each BVDV species is divided into many genotypes [4], which are further divided into two biotypes, cytopathogenic and non-cytopathogenic, based on their pathogenic properties in certain cultured cells [5]. Intrauterine infection with non-cytopathogenic BVDV during bovine gestation results in the production of persistently infected (PI) calves [6], and PI cattle shed large amounts of the virus throughout their lives because of immune tolerance to BVDV, representing the main source of infection in herds. Therefore, controlling PI cattle is an important countermeasure to prevent losses from BVD [7].

The most basic countermeasure for controlling the spread of BVDV is conducting routine screening inspections, such as bulk milk tests and spot tests, to search farms containing PI cattle [7]. In general, after the routine inspections, test positive farms are suspected to contain BVDV-infected cattle and then subjected to an inspection of all cattle to identify BVDV-infected individuals; a definitive diagnosis to identify PI animals is performed using antigen detection test such as RT-PCR or enzyme-linked immunosorbent assay (ELISA) for paired sera, and individuals showing test-positive in both sera are defined as PI animals.

During these inspections, an antibody detection test such as virus neutralization (VN) test is also performed to obtain supportive information; for example, VN test results could be used to confirm immune status of herds as well as immune tolerance of PI animals against BVDV. However, information obtained from the result of a single point VN test is insufficient to know the time when individuals around PI animals were infected with the virus and to evaluate what subclasses of immunoglobulin are included in the sera.

IgM antibodies are produced immediately after infection by pathogens, meaning that the presence of IgM antibodies can be evidence for a recent infection without paired sera. Hence, we hypothesized that detection of BVDV-specific IgM antibodies might be a supportive tool for VN test. However, there is little available information regarding the detection of anti-BVDV IgM antibodies on farms with PI animals. Therefore, in this study, we aimed to evaluate whether BVDV-specific IgM antibodies were detectable on farms at the point of PI identification and to find characteristics of herds in which IgM-positive animals were identified. We constructed a BVDV-specific IgM capture ELISA using recombinant E2 antigen and attempted to detect anti-BVDV IgM antibodies in clinical samples collected from farms on which PI cattle were identified.

2. Materials and methods

2.1. Cells and viruses

MDBK cells were maintained in Eagle's medium (EMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5 % heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan). 293T cells (ATCC CRL-3216) were maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) supplemented with 10 % FBS along with 100 units/mL penicillin and 100 μg/mL streptomycin.

BVDV1a Nose and BVDV2a KZ91CP strains were used for VN tests [8]. BVDV1b Gunma/15/19 strain (GenBank accession no. LC630469), which was isolated from PI cattle in Japan [9], was used for the infection experiment. All virus was propagated in MDBK cells.

2.2. Clinical samples

Clinical serum samples used in this study were collected as a result of clinical inspection at Livestock Hygiene Service Centers in Oita Prefecture, Japan. According to the interview with farm owners, vaccination history of each individual was unclear due to their varying migration histories. Serum samples obtained from 12 cattle, which had been raised in National Institute of Animal Health under high biosecurity condition and confirmed as BVDV antigen negative, were kindly provided by Dr. Shogo Higaki (National Institute of Animal Health, National Agriculture and Food Research Organization). Immune sera against BVDV were kindly provided by Dr. Ken-Ichiro Kameyama (National Institute of Animal Health, National Agriculture and Food Research Organization); VN titers of anti-BVDV1a Nose strain (#9702 and #9836) and anti-BVDV2a KZ91CP strain (#9721 and #9873) sera against homologous strains were 7.0 and 9.0, and 10.0 and 10.0, respectively.

2.3. VN test

Fifty microliters of sera was subjected to two-fold serial dilution in the growth medium in 96-well plates, and 50 μL of the indicator virus was added at 200 TCID₅₀ per well, and the plates were incubated for 1 h at 37 °C. The serum-treated samples were then inoculated to 100 μL of MDBK cells (approximately 2 × 10⁵ cells/well), and the mixtures were incubated at 37 °C in a 5 % CO2 incubator. Cells were observed for cytopathic effects (CPE) at 1 week post infection. The test was performed in duplicate, and the VN titer was defined as the reciprocal of the highest dilution at which no CPE was observed in both wells. VN titers are provided as logarithm of 2 in this manuscript.

2.4. Expression plasmid construction

A segment of BVDV E2 cDNA was amplified from genome RNA derived from BVDV1a Nose strain (position 3360 to 4391 in GenBank accession no. AB078951.1); viral RNA was purified using a QIAamp Viral RNA Mini kit (QIAGEN, Tokyo, Japan), followed by RT-PCR using a PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa, Shiga, Japan) with primers no. 1 and 2 (Supplemental Table 1). The PCR conditions were as follows: 50 °C for 30 min, 94 °C for 2 min, and 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min. The resultant fragment was cloned into the EcoRI and NotI recognition sites of pCAG neo (FUJIFILM Wako, Osaka, Japan) using the HD Cloning Kit (TaKaRa). Next, a partial sequence encoding the extracellular domain of E2 protein was amplified from the cloned E2 sequence to obtain secretory E2 protein; the target sequence was amplified using PrimeSTAR Max DNA Polymerase (TaKaRa) with primers no. 1 and 3 (Supplemental Table 1). The PCR conditions were as follows: 35 cycles of 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 2 min. The resultant fragment was cloned into the EcoRI and NotI recognition sites of pCAG neo (FUJIFILM Wako) using the HD Cloning Kit (TaKaRa). During the cloning procedure, DNA sequences encoding a signal sequence of IgG derived from pDisplay (Thermo Fisher Scientific) and a synthesized his-tag peptide encoding sequence was attached to the secretory E2 construct (Supplemental Figs. 1A and 1B). The constructed plasmid was designated as pGAG-E2-ΔTM.

2.5. Preparation of recombinant proteins

pGAG-E2-ΔTM was transfected into 293T cells using polyethylenimine (PEI) in accordance with our previous report [10]. Briefly, 30 μg of the plasmid was mixed with OPTI-MEM (Thermo Fisher Scientific) containing 240 μL of PEI reagent (2 mg/mL concentration of PEI MAX MW 40,000; Polysciences, Warrington, PA, USA), and the mixture was then transfected into 293T cells grown to confluency in T225 cell culture flasks. The supernatant of the transfected cells was harvested at 4 days post-transfection and subjected to a purification procedure; the recombinant protein was purified using Ni sepharose (GE Healthcare, Tokyo, Japan) in accordance with the manufacturer's protocol. After the purification step, the recombinant protein was suspended in PBS and stored at −80 °C until the following experiments. An empty pCAG plasmid was used for preparing mock antigen in the same manner.

Recombinant E2 protein derived from the baculovirus expression system was prepared by KAICO Ltd. (http://www.kaicoltd.jp/english-home/); briefly, the constructed secretory E2 sequence was cloned into a recombinant baculovirus, followed by injection to silkworms in accordance with the previous report [11]. The recombinant protein was purified using Ni sepharose (GE Healthcare) in the same manner as for the 293T cell-derived protein. As a mock antigen, baculovirus with empty vector was used in the same manner.

2.6. SDS-PAGE and western blotting (WB)

Purified recombinant proteins were mixed with 4 × Laemmli sample buffer (BIO-RAD, Hercules, CA, USA) containing 200 mM dithiothreitol (DTT) and boiled for 5 min at 95 °C. The proteins were separated on 5–20 % polyacrylamide gradient gels (ATTO, Tokyo, Japan) and transferred to polyvinylidene difluoride (PVDF) membranes using the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). The membranes were then incubated in 5 % skim milk (FUJIFILM Wako) in T-PBS buffer (PBS containing 0.05 % Tween 20) at room temperature for 1 h. After the blocking step, the membranes were incubated with mouse anti-His tag MAb (D291-3; MBL, Tokyo, Japan) in 5 % skim milk in T-PBS buffer at room temperature for 1 h. After the washing step, the membranes were incubated with peroxidase-conjugated goat anti-mouse IgG MAb (ab6789; Abcam, Cambridge, UK) in 5 % skim milk in T-PBS buffer at room temperature for 1 h. The detected proteins were visualized with Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) or TMB substrate (Sigma-Aldrich).

2.7. Experimental infection

Two healthy cattle (Japanese Black, female, 55–65 months of age) were introduced from the research farm of the National Agriculture and Food Research Organization and were inoculated intranasally with BVDV1b Gunma/15/19 strain in 4 ml of culture medium (10⁶ TCID₅₀ per head). Serum samples were collected every week until one month after virus inoculation. The animal experiment was carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Animal Health, National Agriculture and Food Research Organization, with ethical approval (authorization number: 21–053).

2.8. ELISA

2.8.1. BVDV-specific antigen-detection ELISA

An IDEXX BVDV Ag/Serum Plus Test kit (IDEXX, Westbrook, ME, USA) was used for detecting viral antigen in sample sera. All procedures were performed in accordance with the manufacturer's protocol, and the results are shown as S–N values. The cut-off value of the BVDV Ag/Serum Plus Test kit is S–N value 0.30.

2.8.2. BVDV-specific IgG-detection ELISA

To detect BVDV-specific IgG antibodies, baculovirus-derived purified E2 antigen was diluted at a concentration of 1 ng/μL in PBS, and 50 μL of the E2 antigen solution was added to the 96-well microplates (Maxisorp; Nunc, Denmark). After incubation overnight at 4 °C, the plates were washed three times with T-PBS, and 300 μL T-PBS containing 5 % skim milk was added, followed by incubation at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of 100-fold diluted immune sera was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of peroxidase-conjugated polyclonal goat anti-bovine IgG antibody (101-035-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 100 μL of TMB substrate (Sigma-Aldrich) was dispensed to each well, and the plates were incubated at 37 °C for 30 min. The enzymatic reaction was stopped by the addition of 100 μL 1 M sulfuric acid, and the absorbance was measured using a spectrophotometer (TECAN, Männedorf, Switzerland) with a 450 nm filter.

2.8.3. BVDV-specific IgM-detection ELISA

To detect BVDV-specific IgM antibodies (Supplemental Fig. 1E), polyclonal sheep anti-bovine IgM antibody (NB758; Novus Biologicals, Centennial, CO, USA) was diluted at 10 ng/μL in 0.1 M carbonate buffer, and 50 μL of the capture antibody solution was added to the 96-well microplates (Maxisorp; Nunc). After incubation overnight at 4 °C, the plates were washed three times with T-PBS, and 300 μL of T-PBS containing 5 % skim milk was added, followed by incubation at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of 10-fold diluted sample sera was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of the purified E2 antigen diluted at a concentration of 1 ng/μL was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of mouse anti-His tag MAb (D291-3; MBL) was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 50 μL of peroxidase-conjugated polyclonal goat anti-mouse IgG MAb (ab6789; Abcam) was added to each well, and the plates were incubated at 37 °C for 1 h. After washing three times with T-PBS, 100 μL of TMB substrate (Sigma-Aldrich) was dispensed to each well, and the plates were incubated at 37 °C for 30 min. The enzymatic reaction was stopped by the addition of 100 μL 1 M sulfuric acid, and the absorbance was measured using a spectrophotometer (TECAN) with a 450 nm filter. Serum collected from cattle no. 1 at 2 weeks post experimental infection was used as a positive control for measuring clinical samples.

2.9. Genotyping of BVDV isolated from clinical samples

Genotyping of BVDV isolated from clinical samples was carried out in accordance with the previous report [9]; briefly, 5′-untranslated region (5′-UTR) nucleotide sequences were amplified by RT-PCR and sequenced, followed by phylogenetic analysis with reference strains and recent Japanese isolates.

3. Results

3.1. Preparation of recombinant E2 protein

The extracellular domain of the E2 protein derived from BVDV1a Nose strain was constructed and applied to the expression analysis using 293T cells to confirm whether the designed antigen was obtained as a secreted soluble protein. SDS-PAGE followed by WB analysis showed that a clear band of approximately 50 kDa was observed only in the pGAG-E2-ΔTM-transfected sample, suggesting that the designed antigen could be obtained as a secreted soluble protein (Supplemental Fig. 1C).

Since the designed antigen was obtained as the secreted soluble protein, we then confirmed whether the baculovirus-derived system could be a substitute for the 293T cell-derived antigen, as the baculovirus-derived expression system is more cost-effective and high-yield system than mammalian cell expression system. The baculovirus-derived E2 protein showed similar results to the 293T cell-derived protein by SDS-PAGE and WB analysis; a band of approximately 50 kDa was observed after purification (Supplemental Fig. 1D).

To confirm the antigenicity of the baculovirus-derived protein, we performed BVDV-specific IgG ELISA in which the purified sample solutions were coated on the plate and subsequently reacted with anti-BVDV immune sera. The result of the IgG ELISA showed that the recombinant E2 protein clearly reacted with both anti-BVDV1a (Nose strain) and -BVDV2a (KZ91CP strain) immune sera, whereas the mock antigen did not (Fig. 1). The OD values against BVDV1a tended to be greater than those against BVDV2a, and anti-BVDV2a immune serum #9721 slightly reacted with mock antigen.

Fig. 1.

Detection of BVDV-specific IgG antibody by ELISA. Baculovirus-derived recombinant E2 and mock antigens were coated on the plate, followed by reaction with anti-BVDV immune sera. Data are presented as the mean ± standard deviation (n = 3). White and grey bars indicate results from recombinant E2 and mock antigens, respectively, and t-test was performed for statistical analysis.

3.2. Evaluation of the BVDV-specific IgM ELISA

To confirm the detection period of anti-BVDV IgM antibodies, sera obtained from two cattle that were experimentally infected with BVDV1b were used for detecting BVDV-specific IgM antibodies. BVDV1-specific VN titers and IgG ELISA values were raised at 2 weeks post infection and were elevated up to 4 weeks in both cattle, whereas IgM ELISA values increased at 2–3 weeks post infection and tended to decrease at 4 weeks (Fig. 2). It should be noted that the sera obtained from cattle no. 2 non-specifically reacted with the mock antigen in both IgG and IgM ELISA, resulting in a reduction of the S–N contrast (Supplemental Fig. 2).

Fig. 2.

Transition of VN titer (A), BVDV-specific IgG (B) and IgM (C) antibody in sera obtained from the infection experiment. Two cattle were infected with BVDV1b, and sera were sequentially collected once a week post infection for four weeks. Anti-BVDV1 VN titer is shown as logarithm of 2, and ELISA value was calculated by subtracting the OD450 value of the mock antigen from that of E2.

3.3. Determination of cut-off value of the IgM ELISA

To determine the cut-off value of the IgM ELISA for differentiating BVDV-infected and -uninfected cattle, sera obtained from 12 healthy cattle were subjected to the assay. These 12 animals were negative for BVDV antigen detection test, but 10 out of 12 animals showed high VN titers against both BVDV1 and BVDV2 as the result of previous infection (Supplemental Fig. 3). On the basis of the results of IgM ELISA from these cattle, a cut-off value of 0.88 was determined as follows; the ratio of sample and positive control (S/P) was calculated after subtraction of the OD450 value of the mock antigen from that of E2, and the threshold was calculated as average + 3SD (Supplemental Fig. 3).

3.4. Evaluation of clinical samples using the constructed IgM ELISA

3.4.1. Evaluation of clinical samples on farm 1

On farm 1, 124 cattle were separately raised in 13 barns based on their age (Fig. 3A). Cattle over 20 months of age were raised in barns A, B, E, and F, while cattle under 18 months of age were raised in barns C, D, G, H, I, J, K, L, and M; specifically, calves under 11 months of age were raised in barns H, I, J, K, L, and M. On farm 1, inspection of all individuals was performed because a certain animal showed diarrhea with BVDV infection. As a result of the inspection, one animal each in barns C and G were positive for the antigen detection ELISA, and the OD value of the animal in barn C was higher than that in barn G (Fig. 3B). Subsequently, the animal in barn C was definitively diagnosed as persistently infected with BVDV by the second inspection, whereas the animal in barn G was diagnosed as an acute infection. Sequence analysis showed that the detected virus was the BVDV1b subgenotype. Conventional VN test showed that all adult cattle in barns A, B, E, and F showed high antibody titers against BVDV1 (mean ± SD of antibody titer was 6.0 ± 0.0, 8.3 ± 1.5, 8.0 ± 0.0, and 8.3 ± 1.2, respectively) (Fig. 3C). On the other hand, young cattle in barns D, H, I, J, K, L, and M showed antibody titers of various values against BVDV1 (mean ± SD of antibody titer was 2.0 ± 0.0, 6.0 ± 2.7, 0.0 ± 0.0, 1.3 ± 3.0, 3.0 ± 1.7, 7.3 ± 2.6, and 7.5 ± 1.9, respectively); individuals showing high antibody titers were considered to be colostrum-derived, since most of these cattle were under 6 months of age and the antibody titer showed clear negative-correlation with age (Supplemental Fig. 4). It was noteworthy that calves nos. 43, 104, and 111 in barns D, J, and K showed higher VN titer than the estimated line, implying that these animals had already been infected with BVDV before the inspection (Supplemental Fig. 4). Regarding the BVDV-positive barns (C and G), the distribution of antibody titers differed from each other, although the age of cattle in these barns was similar; all cattle in barn C showed high anti-BVDV1 titers (mean ± SD of antibody titer was 7.9 ± 2.6), whereas sero-positive and -negative cattle coexisted in barn G (mean ± SD of antibody titer was 3.5 ± 4.1) (Fig. 3C). The antibody titers against BVDV2 were lower than those of BVDV1, although the VN titer magnitude appeared to be correlated (Supplemental Fig. 5A, R² = 0.58); the distribution of antibody titers against BVDV1 and BVDV2 were 1.0–12.0 and 1.0–6.0, respectively (Fig. 3C, Supplemental Fig. 5A). In this situation, one individual each in barns B and C and three cattle in barn G were positive for BVDV-specific IgM ELISA (Fig. 3D).

Fig. 3.

Measurement values of BVDV inspection for all individuals on farm 1.

(A) Age of cattle raised in each barn. Letters on the x-axis indicate barns on the farm.

(B) Detection of BVDV-specific antigen by ELISA. ELISA value was calculated by subtracting the OD450 value of the negative control from that of the sample. ELISA value of 0.30 (dashed line) shows the cut-off line.

(C) VN titers against BVDV1 and BVDV2. Y-axis is shown as logarithm of 2.

(D) Detection of BVDV-specific IgM antibody by ELISA. The S/P value was calculated as follows: ratio of sample and positive control (S/P) was calculated after subtraction of OD450 value of the mock antigen from that of E2. S/P value of 0.88 (dashed line) shows the cut-off line.

3.4.2. Evaluation of clinical samples on farm 2

On farm 2, 246 dairy cattle were being raised, and the cattle were separately annotated into four stages: calves, heifers, milking, and dry cows. The inspection of all individuals was performed because a routine inspection using bulk tank milk showed positive results for BVDV detection RT-PCR. As a result of inspecting all cattle, one calf and two cattle in each of the milking and dry stages were positive for the antigen detection ELISA (Fig. 4A). These 5 cattle were definitively diagnosed as PI animals by the second inspection, and sequence analysis showed that the detected virus was the BVDV1b subgenotype. Conventional VN tests showed that the antibody titer against BVDV1 was higher than BVDV2 in all stages; the distribution of VN titer was 1.0–12.0 against BVDV1 and 1.0–7.0 against BVDV2, respectively (Fig. 4B, Supplemental Fig. 5B). Regarding the anti-BVDV1 antibody, all cattle except for the PI animals in the heifer, milking, and dry stages showed high antibody titers (mean ± SD of antibody titer was 7.8 ± 1.6, 8.0 ± 1.9, and 7.9 ± 3.1, respectively), but the antibody titer levels of calves varied; anti-BVDV1 antibody titers of calves were distributed from negative through 11.0 (mean ± SD of antibody titer was 5.6 ± 2.6) (Fig. 4B). In this situation, two calves were positive for the BVDV-specific IgM ELISA, and one of them showed an especially high S/P value (Fig. 4C).

Fig. 4.

Measurement values of BVDV inspection for all individuals on farm 2.

(A) Detection of BVDV-specific antigen by ELISA. ELISA value was calculated by subtracting the OD450 value of the negative control from that of the sample. X-axis indicates the cattle rearing stages on the farm. ELISA value of 0.30 (dashed line) shows the cut-off line.

(B) VN titers against BVDV1 and BVDV2. Y-axis is shown as logarithm of 2.

(C) Detection of BVDV-specific IgM antibody by ELISA. The S/P value was calculated as follows: ratio of sample and positive control (S/P) was calculated by subtracting the OD450 value of the mock antigen from that of E2. The S/P value of 0.88 (dashed line) shows the cut-off line.

4. Discussion

We herein constructed the BVDV-specific IgM capture ELISA using recombinant E2 antigen and showed that BVDV specific IgM antibody were detected in clinical samples collected from farms with identified PI cattle. The constructed IgM ELISA could detect anti-BVDV IgM antibodies during approximately 2–3 weeks post infection in our infection experiment. Several IgM-positive cattle were identified from clinical samples, and 5 out of 124 and 2 out of 246 animals were positive for the IgM ELISA on farm 1 and 2, respectively.

Summarizing the results of clinical samples, IgM-positive animals tended to be identified in relatively young animals showing sparse VN titers (barn G on farm 1, and calves on farm 2). On farm 1, one PI and one acutely infected animal were identified in barns C and G, and VN titers of the herd in barn C were uniformly high, while those in barn G varied. This result implied that BVDV had spread from the PI animal in barn C and that the BVDV infection was ongoing in barn G. In fact, three cattle in barn G were positive for IgM ELISA, demonstrating that these cattle might be recently infected with BVDV. On the other hand, on farm 2, five PI cattle were identified among the calves, dry and milking cows, and all cattle except for the calves showed uniformly high VN titers against BVDV1. These results implied that BVDV might have already spread throughout the farm, except for some of the calves. Consistent with this interpretation, two calves were positive for IgM ELISA, meaning that these calves might be recently infected with BVDV. Regarding the low positive rate for the IgM ELISA on these farms, it was highly possible that these herds had already been infected with BVDV derived from PI animals a long time ago, resulting in disappearance of IgM antibodies. The data that almost all adults were solidly seropositive was consistent with this interpretation, although their vaccination history was unclear.

There are some limitations in this study. First, we did not evaluate whether the BVDV genotype affected the results of the IgM ELISA. The recombinant E2 antigen used in this study was derived from BVDV1a Nose strain, which is the standard indicator virus for the VN test in Japan. Our group previously reported that recent popular genotypes in Japan were BVDV1b and 2a [9], meaning that the BVDV1a-derived antigen might have a risk of antigenic mismatches [8,12]. Although we confirmed that the prepared E2 antigen clearly reacted with both immune sera against BVDV1a and 2a as well as clinical samples infected with BVDV1b, the effect of antigen mismatch should be carefully considered. Second, the sensitivity of the constructed IgM ELISA was low, and the method had a risk of false-negatives. As shown in Supplemental Fig. 3, we set the S/P cut-off value at 0.88 to cover 99 % of negative samples. However, two negative specimens used in this study showed a non-specific reaction against the mock antigen, resulting in high S/P values (Supplemental Fig. 3). Hence, these high S/P values made the cut-off value slightly high. Regarding the non-specific reaction, blood samples collected from cattle no. 2 in the infection experiment also nonspecifically reacted with the mock antigen (Supplemental Fig. 2); however, we were unable to determine the cause of this nonspecific reaction. Thus, the nonspecific reaction affected the sensitivity of the IgM ELISA, and it was possible that some of clinical samples were false-negative for the IgM ELISA. Further improvement is required to utilize the IgM detection assay.

In conclusion, this study demonstrated that BVDV-specific IgM antibodies were detectable on farms at the point of PI identification and that IgM-positive animals tended to be identified in relatively young animals showing sparse VN titers. Although the number of cases are limited and some issues need to be resolved, these findings would contribute to interpret a status of farms on which PI cattle were identified.

Funding

This study was funded by the Japan Association for Livestock New Technology Research Grant FY2022 (to KA) and partially supported by the research project on “Regulatory research projects for food safety, animal health, and plant protection (grant JPJ008617.23812859)” funded by the Ministry of Agriculture, Forestry, and Fisheries of Japan (to KA, AN, and YM).

Ethics approval statement

All clinical samples used in this study were collected in the course of clinical inspections at Livestock Hygiene Service Centers in Oita Prefecture, Japan, and all procedures were performed in accordance with the local government regulations. Animal experiment was carried out in strict accordance with local guidelines and with ethical approval from the National Institute of Animal Health, National Agriculture and Food Research Organization (authorization number: 21–053).

CRediT authorship contribution statement

Kiyohiko Andoh: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft. Takumi Hayashi: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. Asami Nishimori: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Writing – review & editing. Yuichi Matsuura: Data curation, Methodology, Resources, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the supplementary data to this article.