The wild-type BoHV-1 HB06 strain (GenBank accession number: AJ004801.1) was maintained in the National Key Laboratory of Agricultural Microbiology. Madin-Darby bovine kidney cells (MDBK) were procured from the China Institute of Veterinary Drug Control and used in this study.

The BoHV-1 HB06 strain was cultured as previously described (Marawan et al., 2021). Briefly, the BoHV-1 HB06 wild-type strain was previously isolated from clinical samples by our laboratory, and the virus was propagated in Madin-Darby bovine kidney (MDBK) cells using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Inner Mongolia Opcel Biotechnology Co., Ltd., Hohhot, China). The cell culture was maintained at 37°C in a 5% CO₂ incubator.

A total of 15 Holstein dairy calves, aged between two to four months, were included in the study. All calves in our experiment came from the same pasture, and the calves were isolated for a week before the experiment began to minimize the stress of the transfer in the experiment and to ensure the stability of the calve microbiome at the beginning of the experiment. These calves were purchased from pasture and tested seronegative for M. bovis, BoHV-1, Pasteurella and Mannheimia haemolytica. The calves were divided into 5 groups.

All cattle were housed in isolation to prevent cross-infection. The treatment groups were as follows:

The calves nostrils are cleaned and sterilized before we collect the nasal swab samples. The swabs we use are individually wrapped and sterile, and we collect the samples to ensure that the swab goes 15–20 cm deep into the calves nasal cavity. Nasal drops and no additional treatment samples were collected daily for 28 days following immunization. The samples were stored at −80°C for subsequent quantitative PCR (qPCR) analysis and 16S rRNA gene sequencing.

Procoagulant tubes contains thrombin and cabosil were used to collect blood samples from the jugular vein. Blood samples were collected weekly throughout the duration of the experiment. On top of that, blood samples were also collected on days 1, 3, and 5 after immunization for additional analysis.

Microbial genomic DNA was extracted from nasal swabs collected on the day of immunization and on the 7th day after immunization. The DNA extraction was performed using the E.Z.N.A.® Tissue DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The quality and concentration of the extracted DNA were assessed by 1.0% agarose gel electrophoresis and a NanoDrop2000 spectrophotometer (Thermo Scientific, United States).

The hypervariable region V3-V4 of the bacterial 16 s rRNA gene were amplified with primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5’-GGACTACHVGGGTWTCTAAT-3′) in a T100 Thermal Cycler PCR thermocycler (BIO-RAD, USA). The PCR product was extracted from a 2% agarose gel and purified using the PCR Clean-Up Kit (YuHua, Shanghai, China) according to the manufacturer’s instructions. The purified amplicons were quantified using Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA).

The purified amplicons were pooled in equimolar amounts and subjected to paired-end sequenced on an Illumina PE300/PE250 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Raw FASTQ files from the sequencing data were de-multiplexed using an in-house Perl script. The sequences were then quality-filtered using fastp version 0.19.6 and merged using FLASH version 1.2.7.

The optimized sequences were clustered into operational taxonomic units (OTUs) using UPARSE 7.1 with a 97% sequence similarity level. The most abundant sequence for each OTU was selected as a representative sequence.

To minimize the effects of sequencing depth on alpha and beta diversity measure, the number of 16S rRNA gene sequences from each sample were rarefied to 56,182, which still yielded an average Good’s coverage of 99.75%.

The taxonomy of each OTU representative sequence was analyzed using the RDP Classifier version 2.2 against the 16S rRNA gene database (eg. Silva v138) with a confidence threshold of 0.7.

Alpha diversity indices, such as observed OTUs, Chao1 richness, Shannon’s index, and Good’s coverage, were calculated using Mothur v1.30.1 based on OTUs information.

qPCR was used to quantify the abundance of the following BRD-associated pathogenic bacteria in the collected nasal swab samples: Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Trueperella pyogenes and Mycoplasma bovis (wild-type strain). The primers and probes used for the qPCR analysis are detailed in Table 1.

Serum antibodies against M. bovis were detected using a competitive enzyme-linked immunosorbent assay (ELISA) as previously described (Zhang et al., 2024). Briefly, the test serum samples were diluted fourfold, and then added along with HRP-labeled monoclonal antibodies to an M. bovis p579 protein-coated plate. Positive and negative serum controls were also included. The plate was incubated at 37°C for 60 min. After washing, 100 μL of substrate chromogenic solution was added to each well and incubated at room temperature, away from light, for 10 min. The OD_450nm value was immediately read after stopping the reaction.

The blocking rate (PI value) was calculated as follows:

Blocking rate = 1 - (Sample OD_450nm/Mean OD_450nm of negative control serum).

Conditions for the establishing the test:

0.65 < OD_450nm negative control <2.0.

PI value of positive control >0.6.

PI value of sample ≥ 0.41 indicates a positive result.

PI value of sample < 0.41 indicates a negative result.

Serum samples were first heat-inactivated at 56°C for 30 min. The inactivated serum was then serially diluted in a 96-well cell culture plate. Next, 100 TCID₅₀ of BoHV-1 HB06 virus was incubated with the diluted serum samples at 37°C in a 5% CO₂ incubator for 1 h. This allowed any neutralizing antibodies in the serum to bind to the virus. The serum-virus mixture was then transferred to a 96-well cell culture plate containing MDBK cells. The plate was cultured in a 5% CO₂ incubator at 37°C for three days. The Neutralizing antibody titers were calculated using the Reed-Muench method. This method determines the highest serum dilutions that successfully inhibits BoHV-1 infection of the MDBK cells.

Serum levels of Cortisol (COR) superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using commercial kits. Specifically, serum COR levels were measured using commercial ELISA kits (Meimian Industrial Co., Ltd. Jiangsu, China), while serum SOD, GSH-Px and MDA levels were measured using kits from Beyotime Biotechnology (Shanghai, China). All measurements were performed according to the manufacturer’s instructions.

The animal experiment protocol was approved by the Animal Experiment Ethics Committee of Huazhong Agricultural University (Huazhong Agricultural University Ethics Approval Number: HZAUCA-2024-0036) and conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of Wuhan, Hubei, China.

Shapiro–Wilk normality test was used for normal distribution. Statistical analysis was performed using Student’s t-test and one-way analysis variance (ANOVA). Significant differences between groups were determined using the following p-value thresholds: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****). Error bars in the figures represented the standard error of the mean.

To assessed microbiome diversity, 16S rRNA gene sequencing was used. The results showed that after vaccination, the Se/E, APS, CS, VC and Mock group had 844, 1,311, 931, 925 and 1,523 operational taxonomic units (OTUs), respectively.

In the Se/E group, 69.55% (587/844) of the OTUs were shared with the Mock group, and 58.18% (491/844) were shared with the VC group. Of these, 53.20% (449/844) were present in all three groups (Figure 1A).

In the APS group, 61.40% (805/1311) of the OTUs were shared with the Mock group, and 48.82% (640/1311) were shared with the VC. Of these, 44.47% (583/1311) were present in all three groups (Figure 1B).

In the CS group, 70.89% (660/931) of the OTUs were shared with the Mock group, and 59.29% (552/931) were shared with the VC group. Of these, 54.67% (509/931) were present in all three groups (Figure 1C).

For the Se/E, APS and CS groups, 466 OTUs were shared, which occupied 55.21% (466/844) of the Se/E group, 35.55% (466/1311) of the APS group and 50.05% (466/931) of the CS group (Figure 1D).

Additionally, ACE, Chao, Sobs, Pielou_e, and Shannon index were used to evaluate the microbiome diversity pre-immunization (day 0) and post-immunization (day 7) (Figures 2A–E). The data showed no statistically significantly difference in these diversity measures among all the treatment groups at both time points, day 0 and day 7. However, the Chao, Sob, Ace indices indicated that microbiome diversity decreased by nearly 50% on day 7 compared to day 0 across the Se/E, APS, CS and only vaccine groups, though this reduction was not statistically significant.

These findings suggested that while the immunization and various supplemental treatments did not significantly alter the overall microbiome diversity, there was a general trend toward reduced diversity approximately one week after immunization, regardless of the treatment group. The lack of statistical significance implies the diversity changes were modest and not drastically impacted by the different interventions.

To further analyze the species composition, the study examined the abundance of the top ten bacteria in the upper respiratory tract. At this level, a total of 32 phyla were identified across all samples, with the dominant phyla being Proteobacteria (39.16% on average), Firmicutes (26.89%), Actinobacteriota (20.64%) and Bacteroidota (11.67), which together accounted for 93.01 to 99.68% of the total (Figure 3A).

At the genus level, a total of 1,003 genera were identified in all samples. The top ten genera in terms of abundance were Moraxella (21.72%), Microbacteriaceae (15.13%), Pasteurella (3.65%), Filobacterium (8.75%), Mycoplasma (7.12%), Mannheimia (3.47%), Streptococcus (4.84%), Corynebacterium (3.01%), Dietzia (1.15%) and Staphylococcus (1.14%) (Figure 3B).

Further analysis showed that the above four dominant phyla did not change significantly before and after immunization across all groups (Figures 4A–D). However, on day 7 post-immunization, the abundance of Pasteurella significantly increased in the VC group compared to day 0 (p < 0.05), and was also significantly higher than the CS (p < 0.05) and Mock groups (p < 0.05) (Figure 5A). Meanwhile, on day 7 post-immunization, the abundance of Mycoplasma in the CS group was significantly increased compared to the pre-immunization period (p < 0.05), and also showed a notable increase compared to all other groups (Figure 5B). Other pathogens showed no significantly difference (Figures 5C–J).

These results indicate that while the overall microbial community structure at the phylum level remained relatively stable, there were some significant shifts in the abundance of specific genera, such as Pasteurella and Mycoplasma, in response to the different immunization and supplementation treatments.

To evaluate the impact of the different treatment regimens on common respiratory pathogens in the calves post-immunization, we detected the shedding of Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Trueperella pyogenes, and Mycoplasma bovis. However, only Pasteurella multocida was successfully detected (Figure 6).

Among all groups, the animals that were vaccinated only with the M. bovis-BoHV-1 vaccine, without any additional treatment, had the highest level of Pasteurella multocida shedding throughout the observation period. On 7 day post immunization, the Pasteurella multocida shedding reached a peak of 10^1.87 copies/μL. Thereafter, the shedding gradually decreased, and by 25 days post-immunization, no Pasteurella multocida shedding was detected in this group.

Se/E group had a lower Pasteurella multocida shedding compared to the vaccinated only group. The shedding in the Se/E group increased to 10^1.52 copies/μL on day 9 post-immunization. It then decreased to 10^0.45 on day 11, and subsequently returned to 10^1.21 copies/μL. After that, the Pasteurella multocida shedding in the Se/E group gradually decreased until day 21 days post-immunization.

The APS group had lower Pasteurella multocida shedding than the Se/E group in the first 5 days post-immunization than. However, the APS group reached a peak of 10^1.2 copies/μL on day 7, two days earlier than the Se/E group. The shedding then decreased to 10^0.53, similar to the Se/E group. But the APS group experienced an increase in shedding again from day 9 to 13, before gradually decreasing. By day 21, no Pasteurella multocida shedding was detected in the APS group, similar to the Se/E group.

The CS group exhibited the lowest Pasteurella multocida shedding among all the treatment groups. Pasteurella multocida shedding was only detected on day 1, 9 and 13, with the levels of 10^0.2, 10^0.29, and 10^0.25 copies/μL, respectively. On all other days, no Pasteurella multocida shedding was detected in the CS group.

These indicated that the addition of all the three supplemental treatments can help reduce the level of Pasteurella multocida shedding in calves compared to the group that was vaccinated only without any other treatment. The antibiotic treatment with CS was the most effective in minimizing Pasteurella multocida shedding, with only sporadic and low-level detection in the CS group compared to the other treatment groups. The timing of the peak Pasteurella multocida shedding varied between the groups, the APS group peaking the earliest ahead of the other treatment groups.

For the COR measurement, the Se/E and APS groups showed almost the same COR levels compared to the Mock group throughout the entire observation period. The CS and VC groups did not differ significantly from each other, but both had extremely higher COR levels compared to the other three groups from day 1 to 7. On day 14, all groups had the same COR level (Figure 7A).

For the SOD measurement, all groups maintained similar levels without significant differences, except on day 5. On day 5, the Se/E and APS groups exhibited a significantly higher SOD levels compared to the VC group (Figure 7B).

For the GSH-Px measurement, the Se/E groups exhibited an extremely significantly higher level compared to VC group on day 3 and 7. In contrast, the other groups maintained relatively stable levels throughout the whole observation, without any significant differences among them (Figure 7C).

For the MDA measurement, all groups maintained similar levels, except on day 5. On day 5, the VC groups exhibited a significantly higher level compared to the Se/E and Mock groups. Additionally, while not statistically significant, the CS had a notable higher MDA level compared to the Se/E, APS and Mock groups, especially on day 5 (Figure 7D).

These results indicated that the Se/E and, to a lesser extent, the APS groups were able to better preserve the antioxidant status and regulate the stress response. And CS intervention was not effective in mitigating the negative impacts of the stressors experienced by the animals.