Multiplex RT-qPCR Application in Early Detection of Bovine Respiratory Disease in Healthy Calves

Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.

In general, several potentially BRD-associated bacterial pathogens are asymptomatic and present in the nasal cavity of cattle, of which the most common pathogens are Mh and Pm [12]. In healthy cattle, a delicate balance exists between these potentially bacterial pathogens and the indigenous microflora of the upper respiratory tract, and moreover, the primary immune mechanism actively prevents the establishment of pathogens into the lower respiratory tract. However, BRD occurs because this homeostatic balance in the upper respiratory tract is disrupted, and pathogens invade the lower respiratory tract [13].
To detect BRD onset early in cattle herd, it is important to continuously monitor the dynamics of major causative pathogens [14]. There are many previous reports concerning the involvement of various pathogens in BRD by testing with PCR and real-time PCR using nasal swabs from infected cattle [15,16]. Furthermore, various investigations based on comparison between nasal swabs from healthy and BRD-infected cattle have been conducted to elucidate a mechanism of BRD onset by pathogens [17][18][19]. To our knowledge, however, there are few reports that monitor the causative pathogens, a sign of BRD onset in healthy cattle for a long period.
The detection of genes from pathogen by a PCR is rapid and useful, suggesting the association of specific pathogens with the disease [20,21]. However, further tests are needed, including measuring antibody titer and monitoring the dynamics of virus genes, to confirm whether an animal is actually infected with the pathogen [22,23]. In addition, we need to fully understand the existence of pathogens in the nasal cavity of healthy cattle [24][25][26].
In this study, we attempted to detect BRD onset early in herds by analyzing the presence of BRD-associated pathogens in nasal swabs and their relationship to BRD onset in healthy cattle with a multiplex real-time RT-PCR (RT-qPCR). The data presented in this study will provide useful information around early detection and mechanism elucidation of BRD onset.

Sampling from Clinically Healthy Calves
Samples from 63 calves maintained in seven farms (4 dairies and 3 nurseries) in Iwate prefecture from 2019 to 2021 are summarized in Supplementary Table S1. Three calves with good health condition per farm were selected for the first time every year and were followed continuously for one year, and then they were changed each year. Nasal swabs and sera were collected four times (June, August, September, and November) per year. Finally, a total of 232 nasal swabs and sera were collected from 20 sample collections. The health conditions of the selected calves during the investigation period were interviewed with each farmer. If those calves had a symptom during the period, they were diagnosed by veterinarians with experiences and knowledge.
For Kakegawa strain (BCoV) and Aichi strain (BToV), 50 µL (200 TCID 50 /0.1 mL per each virus) of these strains was added to well and incubated at 37 • C for 1 h. Thereafter, 100 µL of HRT-18G cells (approximately 1.5 × 10 4 cells) was added into all of the wells and incubated at 37 • C in 5% CO 2 for 7 days.
The appearance of the cytopathogenic effect (CPE) was observed using a microscope (Olympus Corporation, Tokyo, Japan). The VN titer for each serum sample was expressed as the reciprocal of the highest dilution that inhibited CPE.
An antibody titer of less than 2-fold was considered to be negative. A significant increase was defined as a 4-fold or greater increase in an antibody titer compared to two consecutive titers. For calves treated with vaccines containing BVDVs, BAdV7, BRSV, BPIV3, and BHV1, positive results obtained in each pathogen with multiplex RT-qPCR using nasal swabs from the animals, including cohabiting calves, were considered as a significant increase by the field strain or vaccine; otherwise, it was considered to be an effect of the vaccine. Furthermore, because cross-reactivity was observed between BVDV1 and BVDV2 antibodies, the significant increase of BVDV2 antibody titer in BVDV1 vaccinated calves was considered to be due to the vaccine [30,31]. A flowchart combined results by multiplex RT-qPCR with results of antibody titers by VNT is summarized in Figure 1. crease was defined as a 4-fold or greater increase in an antibody titer compared to two consecutive titers. For calves treated with vaccines containing BVDVs, BAdV7, BRSV, BPIV3, and BHV1, positive results obtained in each pathogen with multiplex RT-qPCR using nasal swabs from the animals, including cohabiting calves, were considered as a significant increase by the field strain or vaccine; otherwise, it was considered to be an effect of the vaccine. Furthermore, because cross-reactivity was observed between BVDV1 and BVDV2 antibodies, the significant increase of BVDV2 antibody titer in BVDV1 vaccinated calves was considered to be due to the vaccine [30,31]. A flowchart combined results by multiplex RT-qPCR with results of antibody titers by VNT is summarized in Figure 1.

Figure 1.
A flow chart to evaluate infection status based on results by multiplex RT-qPCR and results by VNT. "No collection of pre-or post-serum" means that the sample was not collected in a session before or after the session in which the result by multiplex RT-qPCR was positive.

Ethics Statement
Clinical samples were collected as a part of routine diagnostic procedures, including monitoring of other pathogens; hence, permission regarding animal ethics was not required.

Confirmation of Respiratory Disease Onset in Clinically Healthy Calves
Throughout interviews with farmers and diagnosis by veterinarians with experiences and knowledge during the investigation period, we confirmed an onset of respiratory disease in seven calves (nos. B1-B3, D4-D6, and G6) and the death of two calves (nos. B1 and G6) from three farms (B, D, and G) (Supplementary Table S1). In addition, there were epidemic cases of respiratory disease on three farms (nos. B, D, and F) during the Figure 1. A flow chart to evaluate infection status based on results by multiplex RT-qPCR and results by VNT. "No collection of pre-or post-serum" means that the sample was not collected in a session before or after the session in which the result by multiplex RT-qPCR was positive.

Ethics Statement
Clinical samples were collected as a part of routine diagnostic procedures, including monitoring of other pathogens; hence, permission regarding animal ethics was not required.

Confirmation of Respiratory Disease Onset in Clinically Healthy Calves
Throughout interviews with farmers and diagnosis by veterinarians with experiences and knowledge during the investigation period, we confirmed an onset of respiratory disease in seven calves (nos. B1-B3, D4-D6, and G6) and the death of two calves (nos. B1 and G6) from three farms (B, D, and G) (Supplementary Table S1). In addition, there were epidemic cases of respiratory disease on three farms (nos. B, D, and F) during the same period, and nine calves (nos. B7-B9, D7-D9, and F4-F6) were suspected to have the disease.

Virus Neutralization
Test Results  (Figure 2). In contrast, the pathogen-specific genes detected by multiplex RT-qPCR using nasal swab samples from 63 clinically healthy calves are shown in Figure 2 and Table 1. The detection rates of PCR-specific genes for 12 pathogens between calves with respiratory symptoms and clinically healthy calves were compared and analyzed using Fisher's exact test [32]. As a result, there were significant differences (p < 0.05) in the detection rates of BCoV, BToV, BRSV, BPIV3, and Mb between the two groups. In contrast, there were no significant differences in the detection rates of BVDVs, BAdVs, BIDV, BHV1, Mh, Pm, and Hs ( Figure 2).

State
In the analysis by multiplex RT-qPCR using 89 calves with respiratory symptoms, 58 samples (65.2%) simultaneously detected two to five pathogens (Supplementary Table S3). In contrast, 102 (44.0%) of 232 samples from 63 clinically healthy calves simultaneously detected two or more pathogens (Supplementary Table S4).

Association of Pathogen Detection by Multiplex RT-qPCR Results with Significant Increase in Antibody Titers before and after Onset of the Disease in 16 Calves with Respiratoty Diseases among 63 Clinically Healthy Calves
In this study, seven calves (B1-B3, D4-D6, and G6) showed respiratory disease and nine calves (B7-B9, D7-D9, and F4-F6) were suspected to have respiratory disease among 63 clinically healthy calves during the investigation period. The multiplex RT-qPCR results from before to after the onset of the disease and the significant increase in antibody titers after the onset of the disease in these 16 calves is summarized in Table 5. The multiplex RT-qPCR results before the onset of disease in ten calves were BAdV (1), Mh (1), Pm (5), Mh and Pm (2), and undetected (1). The multiplex RT-qPCR results at the onset of the disease in eight calves were BCoV, BAdV, Mh, and Pm (1), BCoV, BRSV, Mh, and Pm (1), BCoV, BRSV, and Pm (1), BCoV, Mh, and Pm (1), BCoV and Pm (2), and BRSV and Pm (2). The multiplex RT-qPCR results, after the onset of the disease in 11 calves, were BCoV, BRSV, and Pm (1), BCoV, Mh, and Pm (4), BCoV and Pm (2), Mh and Pm (2), Mh (1), and Pm (1). A significant increase in BCoV antibodies was observed in all 12 calves in which BCoV was detected at the time of onset or after onset. A significant increase in BRSV antibodies was observed in four of five calves in which BRSV was detected at the time of onset or after onset.  S3). In contrast, 102 (44.0%) of 232 samples from 63 clinically healthy calves simultaneously detected two or more pathogens (Supplementary Table S4).

Figure 2.
Comparison of PCR-specific gene detection rates for 12 pathogens by multiplex real-time RT-PCR between 89 nasal swabs from 89 calves with respiratory disease and 232 nasal swabs from 63 clinically healthy calves collected in Iwate prefecture during 2019 to 2021. The black bars indicate the PCR-specific gene detection rate for 12 pathogens from calves with respiratory symptoms, and white bars indicate the PCR-specific gene detection rate for 12 pathogens from clinically healthy calves. The asterisk (*) means significant differences (p < 0.05) between two groups by statistical analysis using Fisher's exact test.

Association of Pathogen Detection by Multiplex RT-qPCR Results with Significant Increase in Antibody Titers before and after Onset of the Disease in 16 Calves with Respiratoty Diseases among 63 Clinically Healthy Calves
In this study, seven calves (B1-B3, D4-D6, and G6) showed respiratory disease and nine calves (B7-B9, D7-D9, and F4-F6) were suspected to have respiratory disease among 63 clinically healthy calves during the investigation period. The multiplex RT-qPCR results from before to after the onset of the disease and the significant increase in antibody titers after the onset of the disease in these 16 calves is summarized in Table 5. The multiplex RT-qPCR results before the onset of disease in ten calves were BAdV (1), Mh (1), Pm (5), Mh and Pm (2), and undetected (1). The multiplex RT-qPCR results at the onset of the disease in eight calves were BCoV, BAdV, Mh, and Pm (1), BCoV, BRSV, Mh, and Pm (1), BCoV, BRSV, and Pm (1), BCoV, Mh, and Pm (1), BCoV and Pm (2), and BRSV and Pm (2). The multiplex RT-qPCR results, after the onset of the disease in 11 calves, were BCoV, BRSV, and Pm (1), BCoV, Mh, and Pm (4), BCoV and Pm (2), Mh and Pm (2), Mh (1), and Pm (1). A significant increase in BCoV antibodies was observed in all 12 calves in which BCoV was detected at the time of onset or after onset. A significant increase in BRSV antibodies was observed in four of five calves in which BRSV was detected at the time of onset or after onset. Figure 2. Comparison of PCR-specific gene detection rates for 12 pathogens by multiplex real-time RT-PCR between 89 nasal swabs from 89 calves with respiratory disease and 232 nasal swabs from 63 clinically healthy calves collected in Iwate prefecture during 2019 to 2021. The black bars indicate the PCR-specific gene detection rate for 12 pathogens from calves with respiratory symptoms, and white bars indicate the PCR-specific gene detection rate for 12 pathogens from clinically healthy calves. The asterisk (*) means significant differences (p < 0.05) between two groups by statistical analysis using Fisher's exact test.

Discussion
Viral and bacterial pathogens play an important role in BRD onset. Therefore, early detection of these pathogens can reduce the damage caused by BRD [33,34]. However, there are numerous pathogens associated with BRD. Therefore, an efficient method to rapidly detect them is needed. For this reason, we used multiplex RT-qPCR to simultaneously detect eight viruses and four bacteria related to BRD [27]. However, it is unclear whether the calf is actually infected with those pathogens in the test by only multiplex RT-qPCR. Hence, we confirmed a history of infection by checking significant increases in antibody titers with VNT. In this study, multiplex RT-qPCR and VNT were performed using nasal swabs or serum samples collected from 63 clinically healthy calves from seven farms and 89 BRD-infected calves from 28 farms in Iwate Prefecture from 2019 to 2021. We attempted to detect BRD-associated pathogens dominant in this region and to understand the roles of the BRD-associated pathogens in clinically healthy calves via these analyses.
To analyze the presence of BRD-associated pathogens in nasal swabs of clinically healthy calves and their relationship to real infection, we compared results between multiplex RT-qPCR and VNT against BVDV1, BVDV2, BCoV, BToV, BAdV, BRSV, BPIV3, and BHV1 using 232 nasal swab samples and 232 serum samples from 69 clinically healthy calves. Herein, we focused on the percentage of samples with a significant increase in antibody titers against BCoV, BToV, BRSV, and BPIV3 that were positive with multiplex RT-qPCR. Except for cases that were unknown by VNT, the pathogens that showed a higher percentage of VNT were BCoV (16 samples with multiplex RT-qPCR positives and significant increase in antibody titers/30 samples with multiplex RT-qPCR positives and 11 samples unknown, therefore 19 samples were adapted: 84.2%), BToV (2/3, 66.7%), and BRSV (2/2, 100%). For BPIV3, there were no applicable samples. Therefore, this suggests that the positive results by multiplex RT-qPCR could be an indicator of real infection. In addition, these findings revealed that several calves with antibody titers of 4 ≤ were shown to be positive by multiplex RT-qPCR. We speculate that those individuals infected more viruses than range that can protect them from BCoV infections by their VN titers. In addition, the positive results might be also influenced by host health condition and co-infection by several pathogens other than the amount of virus exposed.
We compared 12 BRD-related pathogens detected by multiplex RT-qPCR using 89 and 232 nasal swab samples collected from 89 calves with respiratory symptoms and 63 clinically healthy calves during 2019-2021, respectively, owing to finding pathogens that are indicators of BRD onset. As results, we revealed that BCoV, BToV, BRSV, BPIV3, and Mb have been more frequently detected in calves with respiratory symptoms compared to those detected in clinically healthy calves. In previous studies, BRSV, BPIV3, and Mb have been reported as major pathogens of BRD [13,35]. There were several previous reports that BCoV is associated with BRD as well as diarrhea [36,37]. Recently, BToV has also been suggested to be causative virus of BRD [8,9]. Taken together, our data strongly support the previous reports that these pathogens have been closely related to BRD among calves.
Pm and Mh have been reported to be resident bacteria in the nasal cavity [38]. In addition, they are also considered one of the secondary exacerbation factors of BRD. Moreover, it is known that Pm and Mh were further infected in nasal mucosa when damaged by stress and/or viruses, and therefore, symptoms of the infected animals became more severe [39,40]. Our analysis exhibited that there were no significant differences in the detection of Mh-and Pm-specific genes between BRD-infected and healthy calves. Moreover, our data also showed that Mh-and Pm-specific genes were detected several times from the same individuals. Therefore, these findings suggest that Mh and Pm might reside in the nasal cavity of calves and potential exacerbation factors. However, we did not analyze and compare cultivation and quantification of the bacteria between BRD-infected and clinically heathy calves in this study. Thus, we will attempt to perform a further analysis in the future to certify this possibility.
Our analysis found one farm (No.A) where Mh and Pm were never detected for three years. In addition, our data showed that few viruses associated with BRD were detected at the farm. The farm had a small number of cows with little introduction of cattle and no other dairy farms in the surrounding area. These facts suggest that reduction of stress by cattle movement and herd organization, and enhancement of biosecurity in cattle management might be effective for the prevention of BRD [41][42][43].
In this study, we confirmed that seven calves showed BRD symptoms (two calves of them died) out of 63 clinically healthy calves throughout the interview with farmers and diagnosis by veterinarians with experience and knowledge. In addition, there were epidemic cases of respiratory disease on three farms (B, D, and F) during the investigation period, and moreover nine calves (B7-B9, D7-D9, and F4-F6) were suspected to have the disease. Multiplex RT-qPCR and VNT were performed using samples from these 16 calves collected before and after BRD onset. Our analysis revealed one or two pathogens (mainly Mh and Pm) before BRD onset. Subsequently, mixed infections with two to four pathogens of viruses (mainly BCoV and BRSV) and bacteria (mainly Mh and Pm) were detected at BRD onset. Moreover, the number of pathogens detected using a nasal swab sample from healthy and BRD-infected calves was 1.4 and 2.3 pathogens, respectively. The pathogenesis mechanism of BRD has been suggested to be based on stress, immunosuppression, and potentially bacterial pathogens in the nasal cavity. At BRD onset, there is a shift in this homeostatic balance in the upper respiratory tract, resulting in colonization in the lower respiratory tract [12]. Moreover, the virus has been found to play a variety of roles as a stressor, including involvement in immunosuppression, invasion of pathogens into the upper or lower airways, and promotion of secondary infection by the virus [34,44,45]. Our findings might support the pathogenesis mechanism of BRD reported in previous study.
Identifying causative pathogens of BRD requires rapid testing for multiple pathogens, including viruses and bacteria. Our investigation using samples from 16 BRD-infected calves showed that at least BCoV was associated with respiratory symptoms in 12 calves (B7-B9, D4-D9, and F4-F6) on three farms (B, D, and F). Of these, six calves (D4-D9) from farm D were negative for BCoV antibodies before BRD onset and then were infected with BCoV at BRD onset.
In conclusion, we offer the application of multiplex RT-qPCR as a tool to monitor BRD causative pathogens and to detect the risk before BRD is spread within the herds. Analyses by multiplex RT-qPCR using nasal swab samples from calves with respiratory symptoms and clinically healthy calves suggests that BCoV, BToV, BRSV, BPIV3, and Mb are the main causative pathogens of BRD. Moreover, our data revealed that positive results by multiplex RT-qPCR are closely correlated, with significant increases in antibody titers by VNT regarding BCoV, BToV, and BRSV. Therefore, these facts support the idea that continuous monitoring of BRD-associated pathogens in healthy calves using multiplex RT-qPCR would contribute to early detection of BRD. Furthermore, this study found, in one of mechanism of BRD, that Mh and Pm existed in the nasal cavity before BRD onset, and that multiple pathogens of viruses and bacteria, such as BCoV, BRSV, Mh, and Pm were infected there. Therefore, we hope this methodology will be widely spread and accumulate more data in the future.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/v15030669/s1, Table S1: Summary of collection dates, vaccination status, and clinical symptoms from 63 clinically healthy calves, Table S2: List of primer and probe used in multiplex RT-qPCR, Table  S3: Summary of pathogen detections by multiplex RT-qPCR using 89 samples from 89 calves with respiratory symptoms.
Author Contributions: Conceptualization, Y.G. and T.S.; Investigation, Y.G. and K.F.; Methodology, Y.G. and K.F.; Validation, Y.G., K.F. and T.S.; Resources, Y.G. and K.F.; Writing-original draft preparation, Y.G. and K.F.; Writing-review and editing, T.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.