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Pathogenic infection characteristics and risk factors for bovine respiratory disease complex based on the detection of lung pathogens in dead cattle in northeast China

Open AccessPublished:November 01, 2022DOI:https://doi.org/10.3168/jds.2022-21929

      ABSTRACT

      Bovine respiratory disease complex (BRDC) involves multiple pathogens, shows diverse lung lesions, and is a major concern in calves. Pathogens from 160 lung samples of dead cattle from 81 cattle farms in northeast China from 2016 to 2021 were collected to characterize the molecular epidemiology and risk factors of BRDC and to assess the major pathogens involved in bovine suppurative or caseous necrotizing pneumonia. The BRDC was diagnosed by autopsy, pathogen isolation, PCR, or reverse transcription-PCR detection, and gene sequencing. More than 18 species of pathogens, including 491 strains of respiratory pathogens, were detected. The positivity rate of bacteria in the 160 lung samples was 31.77%, including Trueperella pyogenes (9.37%), Pasteurella multocida (8.35%), Histophilus somni (4.48%), Mannheimia haemolytica (2.44%), and other bacteria (7.13%). The positivity rate of Mycoplasma spp. was 38.9%, including M. bovis (7.74%), M. dispar (11.61%), M. bovirhinis (7.94%), M. alkalescens (6.11%), M. arginini (0.81%), and undetermined species (4.68%). Six species of viruses were detected with a positivity rate of 29.33%, including bovine herpesvirus-1 (BoHV-1; 13.25%), bovine respiratory syncytial virus (BRSV; 5.50%), bovine viral diarrhea virus (BVDV; 4.89%), bovine parainfluenza virus type-3 (BPIV-3; 4.28%), bovine parainfluenza virus type-5 (1.22%), and bovine coronavirus (2.24%). Mixed infections among bacteria (73.75%), viruses (50%), and M. bovis (23.75%) were the major features of BRDC in these cattle herds. The risk analysis for multi-pathogen co-infection indicated that BoHV-1 and H. somni; BVDV and M. bovis, P. multocida, T. pyogenes, or Mann. haemolytica; BPIV-3 and M. bovis; BRSV and M. bovis, P. multocida, or T. pyogenes; P. multocida and T. pyogenes; and M. bovis and T. pyogenes or H. somni showed co-infection trends. A survey on molecular epidemiology indicated that the occurrence rate of currently prevalent pathogens in BRDC was 46.15% (6/13) for BoHV-1.2b and 53.85% (7/13) for BoHV-1.2c, 53.3% (8/15) for BVDV-1b and 46.7% (7/15) for BVDV-1d, 29.41% (5/17) for BPIV-3a and 70.59% (12/17) for BPIV-3c, 100% (2/2) for BRSV gene subgroup IX, 91.67% (33/36) for P. multocida serotype A, and 8.33% (3/36) for P. multocida serotype D. Our research discovered new subgenotypes for BoHV-1.2c, BRSV gene subgroup IX, and P. multocida serotype D in China's cattle herds. In the BRDC cases, bovine suppurative or caseous necrotizing pneumonia was highly related to BVDV [odds ratio (OR) = 4.18; 95% confidence interval (95% CI): 1.6–10.7], M. bovis (OR = 2.35; 95% CI: 1.1–4.9), H. somni (OR = 8.2; 95% CI: 2.6–25.5) and T. pyogenes (OR = 13.92; 95% CI: 5.8–33.3). The risk factor analysis found that dairy calves <3 mo and beef calves >3 mo (OR = 5.39; 95% CI: 2.7–10.7) were more susceptible to BRDC. Beef cattle were more susceptible to bovine suppurative or caseous necrotizing pneumonia than dairy cattle (OR = 2.32; 95% CI: 1.2–4.4). These epidemiological data and the new pathogen subgenotypes will be helpful in formulating strategies of control and prevention, developing new vaccines, improving clinical differential diagnosis by necropsy, predicting the most likely pathogen, and justifying antimicrobial use.

      Key words

      INTRODUCTION

      Bovine respiratory disease complex (BRDC) involves a combination of pathogens, stressors, immunologically susceptible animals, and numerous risk factors (Gershwin, 2015;
      • Roland L.
      • Drillich M.
      • Klein-Jöbstl D.
      • Iwersen M.
      Invited review: Influence of climatic conditions on the development, performance, and health of calves.
      ). It is a major cause of morbidity and mortality in feedlot cattle populations and dairy herds, particularly in recently weaned and newly transported calves (
      • Ellis J.A.
      Update on viral pathogenesis in BRD.
      ; Fulton, 2009a;
      • USDA
      Feedlot 2001. Part IV: Health and health management on U.S. feedlots with a capacity of 1,000 or more head. APHIS, National Animal Health Monitoring System.
      ), and it is responsible for severe economic losses in dairy and feedlot herds worldwide. The pathogens associated with BRDC are highly complex. They mainly include viruses, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type-3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine coronavirus (BCoV); and bacteria including Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni; occasionally Trueperella pyogenes, and Mycoplasma spp., particularly M. bovis (
      • Ellis J.A.
      Update on viral pathogenesis in BRD.
      ;
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ;
      • Holman D.B.
      • McAllister T.
      • Topp E.
      • Wright A.D.G.
      • Alexander T.W.
      The nasopharyngeal microbiota of feedlot cattle that develop bovine respiratory disease.
      ). These pathogens can be further divided into multiple genotypes or serotypes. Bovine herpesvirus-1 is divided into 1.1 and 1.2 (a and b); BVDV is divided into type 1 (21 subtypes), type 2 (4 subtypes), and type 3; BPIV-3 is divided into 3a, 3b, and 3c; BRSV is divided into 8 genetic subgroups (I–VIII); and P. multocida is divided into 5 serotypes (A, B, D, E, and F). Mycoplasma spp. belonging to the Mollicutes class include M. mycoides, M. bovis, M. dispar, M. bovirhinis, M. arginini, and M. alkalescens, of which M. mycoides is the most common (Tortorelli and Carrilo Gaeta, 2017;
      • Krešić N.
      • Bedeković T.
      • Brnić D.
      • Šimić I.
      • Lojkić I.
      • Turk N.
      Genetic analysis of bovine respiratory syncytial virus in Croatia.
      ;
      • Zeineldin M.
      • Lowe J.
      • Aldridge B.
      Contribution of the mucosal microbiota to bovine respiratory health.
      ).
      The gold standard for BRDC diagnoses is a pathologic postmortem evaluation immediately after the diagnosis of BRDC but, for ethical and economic reasons, this is usually avoided (
      • Wolfger B.
      • Timsit E.
      • White B.J.
      • Orsel K.
      A systematic review of bovine respiratory disease diagnosis focused on diagnostic confirmation, early detection, and prediction of unfavorable outcomes in feedlot cattle.
      ). A postmortem examination and etiological diagnosis are of great value for diagnosing BRDC but cannot be used for early detection (
      • Caswell J.L.
      • Hewson J.
      • Slavić D.
      • DeLay J.
      • Bateman K.
      Laboratory and postmortem diagnosis of bovine respiratory disease.
      ). The gross lesions in the lungs of cattle with BRDC caused by different pathogens are also diverse. The major types of pneumonia include lobar bronchopneumonia, lobar bronchopneumonia with pleuritis, interstitial pneumonia, broncho interstitial pneumonia, septic pneumonia, pneumonia with embolic foci (bronchopneumonia with multiple foci of caseous necrosis), and suppurative bronchopneumonia (Fulton et al., 2009b;
      • Caswell J.L.
      • Hewson J.
      • Slavić D.
      • DeLay J.
      • Bateman K.
      Laboratory and postmortem diagnosis of bovine respiratory disease.
      ). It is difficult for clinical veterinarians or breeders to distinguish between these types of pneumonia, especially with purulent foci or caseous necrosis foci, called bovine suppurative or caseous necrotizing (BSC) pneumonia or bovine rot pneumonia by clinical veterinarians and breeders.
      At present, pathogen detection in BRDC epidemiological research is mostly limited to detecting pathogens in live animals. The sampling methods commonly include nasal swabs, guarded nasopharyngeal swabs, bronchoalveolar lavage, and transtracheal wash, which cannot fully reflect the pathogens responsible for lung tissue lesions (
      • Timsit E.
      • Holman D.B.
      • Hallewell J.
      • Alexander T.W.
      The nasopharyngeal microbiota in feedlot cattle and its role in respiratory health.
      ;
      • Doyle D.
      • Credille B.
      • Lehenbauer T.W.
      • Berghaus R.
      • Aly S.S.
      • Champagne J.
      • Blanchard P.
      • Crossley B.
      • Berghaus L.
      • Cochran S.
      • Woolums A.
      Agreement among 4 sampling methods to identify respiratory pathogens in dairy calves with acute bovine respiratory disease.
      ;
      • Buczinski S.
      • Pardon B.
      Bovine respiratory disease diagnosis: What progress has been made in clinical diagnosis?.
      ). In addition to traditional bacterial culture, highly sensitive and specific PCR or reverse transcription (RT)-PCR to detect the pathogens based on lung tissues of BRDC cases resulting in spontaneous death or euthanized animals is increasingly used and can accurately reflect the pathogens associated with lung lesions or death (
      • O'Neill R.
      • Mooney J.
      • Connaghan E.
      • Furphy C.
      • Graham D.A.
      Patterns of detection of respiratory viruses in nasal swabs from calves in Ireland: A retrospective study.
      ). The diverse factors that can induce BRDC include transport, mixed groups, climate stress, wet and cold environments, dust, dehydration, hypoxia, toxins, and acute metabolic disorders (
      • Zeineldin M.
      • Lowe J.
      • Aldridge B.
      Contribution of the mucosal microbiota to bovine respiratory health.
      ). However, the relationship between BRDC and risk factors such as climate, age, and breed of susceptible cattle has not yet been reported in China.
      Therefore, the objectives of the present study were to reveal the molecular epidemiological characteristics and risk factors of BRDC and BSC pneumonia in Chinese cattle, and to provide theoretical support for the development of new diagnostic methods and vaccines for the prevention and treatment of BRDC.

      MATERIALS AND METHODS

      Because only postmortem sampling was performed, this study did not require ethical approval.

      PCR Primers

      The PCR primers for detecting bovine respiratory pathogens (Supplemental Table S1; https://doi.org/10.6084/m9.figshare.21341556 ) were synthesized by Shanghai Shenggong Biotechnology Service Co. Ltd. The pathogens related to BRDC selected for detection include viruses [BoHV-1, BVDV, BPIV-3, BRSV, BCoV, and bovine parainfluenza virus type-5 (BPIV-5)], bacteria (P. multocida, Mann. haemolytica, H. somni, and T. pyogenes), and several Mycoplasma spp. using universal primers. The PCR typing primers were used to distinguish the A, B, D, E, and F capsular serotypes of P. multocida. Mycoplasma-specific primers were used to distinguish Mycoplasma mycoides ssp. mycoides small colony (SC) type, M. dispar, M. bovis, M. bovirhinis, M. arginini, and M. alkalescens.

      Samples and Epidemiological Information

      The samples were collected by the Heilongjiang Key Laboratory of Cattle Diseases and the Animal Hospital of Heilongjiang Bayi Agricultural University from July 2016 to July 2021, in northeast China, including Heilongjiang Province, Jilin Province, Liaoning Province, and parts of the Inner Mongolia Autonomous Region bordering Heilongjiang and Jilin Provinces. These samples were derived from 81 cattle herds in 33 counties, as shown in Figure 1 and Supplemental Table S2 ( https://doi.org/10.6084/m9.figshare.21341556 ). The 160 lung samples were collected from sick or euthanized cattle with typical bovine respiratory symptoms (e.g., cough, runny nose, difficulty breathing). As far as possible, the samples were collected aseptically through autopsy within 4 h after death. The lung tissue lesions of cattle that died of BRDC were mostly characterized by consolidation and necrosis or suppuration, and most lesions occur in the cardiac and apical lobes. For each case, 2 pieces of lung tissue (4 to 7 cm) were collected aseptically: one was the lobe with consolidation and the other was the junction of consolidation or purulent foci and nonconsolidated lung tissue. Additionally, necropsy lesions were recorded, and samples with autolysis were discarded. According to the characteristics of lung lesions, BRDC cases were divided into BSC pneumonia and “other” pneumonia. The “BSC pneumonia” category is shown in Supplemental Figures S1 to S5 ( https://doi.org/10.6084/m9.figshare.21341544 ). The visible lesions of the lungs were diverse in BRDC cases, including fibrinous purulent pneumonia (Figure S1a, b), septic pneumonia (Figure S1c, d), abscesses (Figure S1e), suppurative bronchopneumonia (Figure S1f), interstitial pneumonia with necrotic foci (Figure S1g) and caseous necrosis (Figure S1h, i). Pneumonia with pathological lesions of the lungs described above is called BSC pneumonia. The “Other pneumonia” category included visible pulmonary fleshy transformation, interstitial pneumonia, lobar pneumonia, lobular pneumonia, and emphysema. The owner or veterinarian supplied epidemiological information on the diseased cattle (including breed, age, and season), as reported in Supplemental Table S3 ( https://doi.org/10.6084/m9.figshare.21341556 ). Most of the BRDC cases in this study were treated with antibiotics, and the herds had not been administered bovine respiratory disease–related vaccines (BoHV-1, BVDV, BPIV-3, BRSV, BCoV, and BPIV-5, P. multocida, T. pyogenes, Mann. haemolytica, H. somni, and Mycoplasma spp.).
      Figure thumbnail gr1
      Figure 1Spatial distribution and number of bovine respiratory disease complex samples from northeast China.

      Detection of Pathogens in Lung Tissue of Dead Cattle with BRDC

      Pathogens from 160 lung tissue samples from cattle that died from BRDC were detected by PCR or RT-PCR using the primers listed in Supplemental Table S1. The viral pathogens included BoHV-1, BVDV, BPIV-3, BRSV, BCoV, and BPIV-5. The bacterial pathogens included P. multocida, T. pyogenes, Mann. haemolytica, H. somni, and Mycoplasma spp. Mycoplasma spp. samples were further analyzed for M. mycoides ssp. mycoides SC type, M. dispar, M. bovis, M. bovirhinis, M. arginini, and M. alkalescens using species-specific PCR. Lung tissue was also used for routine bacterial isolation.
      The PCR or RT-PCR was performed as follows. Bovine lung samples were collected and ground aseptically. DNA or RNA was prepared using AP-MN-BF-VNA-250 G AxyPrep humoral virus DNA/RNA preparation kit (Axygen 2617) and ReverTra Ace qPCR RT Master Mix (Toyobo) for cDNA synthesis. The reaction volume was 25 μL and included 12.5 μL of Quick Taq HS DyeMix (Toyobo), 2 μL of cDNA or DNA, 1 μL (12.5 pM) of each pathogen-specific sense and antisense primers, and 8.5 μL of sterile deionized water. The cycling parameters were as follows: 5 min after pre-denaturation at 95°C; followed by 35 cycles of 95°C for 1 min, 56°C (62°C for BoHV-1) annealing temperature for 45 s, 72°C extension for 1 min; and a final extension at 72°C for 10 min. Negative and positive controls were included for each PCR, and the PCR products were analyzed using 1% agarose gel electrophoresis.
      During the bacterial test, 2 nutrient agar medium plates with 5% fresh sterile sheep defibrinated blood were inoculated with the aseptically collected lung tissue samples and incubated at 37°C for 48 h under aerobic and CO2 (candle tank method) conditions. The purified bacteria were identified by inspection of bacterial culture characteristics, Gram stain microscopic examination, and identification media (MacConkey and SS medium).
      Bacterial 16S rRNA universal primers were used for PCR amplification and sequencing identification of bacteria that the above methods could not identify.

      Isolation, Identification, and Phylogenetic Analysis of BoHV-1, BVDV, BPIV-3, and BRSV in Lung Tissues

      To reveal the molecular epidemiological characteristics of the viral pathogens of BRDC in China, lung tissues positive for BoHV-1 (55 samples), BVDV (24 samples), and BPIV-3 (21 samples) were used for virus isolation, identification, and genetic evolution analysis.

      Isolation and Identification of Virus

      The aseptically collected lung tissue was mixed with Dulbecco's modified Eagle medium (DMEM) at a ratio of 1:10 and mashed with a tissue masher. The tissue fluid was frozen and thawed twice, centrifuged at 1,000 × g at 4°C for 5 min, and the supernatant was centrifuged at 12,000 × g at 4°C for 15 min. The supernatants were collected, filtered through 0.22-µm filters, and inoculated into Madin-Darby bovine kidney (MDBK) cells at 70 to 80% confluence in a 25-cm2 cell culture flask for 1 h before the supernatants were discarded. This was followed by adding 5 mL of DMEM containing penicillin and streptomycin and incubating the flask at 37°C. The inoculated MDBK cells were observed daily for the appearance of cytopathic effect (CPE). When approximately 60 to 70% CPE was observed in MDBK cells, the cultures were collected and subjected to 3 blind passages in MDBK cells. Virus isolated was detected by PCR using the specific primers shown in Supplemental Table S1.

      Phylogenetic Analysis of BoHV-1, BVDV, BPIV-3, and BRSV

      The partial gC gene (575 bp) of BoHV-1, the partial 5′-UTR (untranslated ragion) sequence (280 bp) of BVDV, the partial M gene (385 bp) of BPIV-3, and the partial G protein gene (848 bp) of BRSV were used as the landmark genes for viral genetic evolutionary analysis (Supplemental Table S1). The genes were amplified by PCR or RT-PCR, and the PCR products were bidirectionally sequenced. Bioedit (version 7.0.5.3; http://www.mbio.ncsu.edu/bioedit/bioedit.html ) was used to assemble and edit the sequences. The BLAST tool of the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi ) was used to compare these sequences with the strain sequences from GenBank. MegAlign software (
      • Thompson J.D.
      • Higgins D.G.
      • Gibson T.J.
      CLUSTAL W: Improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
      ) was used for homology analysis of the isolates' gene sequences and representative reference strains from GenBank. MEGA X software (using the N-J method, Bootstrap was 1000 repeats;
      • Kumar S.
      • Stecher G.
      • Li M.
      • Knyaz C.
      • Tamura K.
      MEGA X: Molecular evolutionary genetics analysis across computing platforms.
      ) was used to construct phylogenetic trees to analyze the genetic variation of isolates.

      Serotyping of P. multocida from Lung Tissues

      To examine the P. multocida serotypes that are currently prevalent in Chinese cattle herds, P. multocida was isolated by culturing lung tissue in 5% fresh sheep blood nutrient agar medium and identified by amplifying part of the kmt1 gene (460 bp) with specific primers. The conserved genes hyaD-hyaC, bcbD, dcbF, ecbJ, and fcbD were amplified by PCR with specific primers to differentiate the P. multocida capsular serotypes A, B, D, E, and F.

      PCR Typing of Mycoplasma spp. in Lung Tissues

      To examine the species and positivity rate of mycoplasmas in infected lung tissues of dead cattle with BRDC, Mycoplasma-positive samples were amplified by PCR using 16S rDNA specific primers of 5 types of mycoplasma, including M. mycoides ssp. mycoides SC, M. dispar, M. bovis, M. bovirhinis, M. arginini, and M. alkalescens.

      Characterization of BRDC Pathogenic Infection

      To explore the characteristics of pathogenic co-infection in BRDC cases, the probability of pathogen appearance was analyzed from cases of multipathogen infection in the 160 BRDC cases. The major pathogens detected in BRDC cattle lungs, including BoHV-1, BVDV, BPIV-3, BRSV, P. multocida, T. pyogenes, Mann. haemolytica, H. somni, and M. bovis were selected for analysis through multiple regression models.
      The lung lesions in the 160 BRDC cases could be classified into 67 cases of BSC pneumonia and 93 cases of “other” pneumonia. To explore the pathogens that played a major role in BSC pneumonia, the number of pathogens detected in the 2 types of bovine pneumonia cases from the 160 cases of BRDC was statistically analyzed.

      Analysis of BRDC Risk Factors

      To explore risk factors related to BRDC in northeast China, we analyzed the effects of age and breed on BRDC. The information was obtained by questioning the owners or attending veterinarians of the 81 cattle herds in which BRDC occurred. Information on sick cattle, including age of birth, breed, month of illness, clinical signs, colostrum management, milk feeding, and transportation, was recorded from the onset of BRDC cases in the herd to the end of the epidemic (including recovery, death, or euthanasia; Supplemental Table S3). The cattle breeds were mainly divided into dairy cattle (Holstein cows) and beef cattle (e.g., Simmental, Charolais, Haifuda, Kobe, Flewich, Australian wagyu).

      Statistical Analysis

      Data were analyzed using GraphPad Prism 5 (GraphPad Inc.) and SPSS Statistics 26 (IBM Corp.) software. The unit of risk factor analysis was herd. The dairy herd size ranged between 500 and 5,000, and the beef herd size was between 50 and 1,000. Noninterpretable results were not included in the analysis. All statistical comparisons were based on a 5% level of significance. Significance was set at P < 0.05, and P < 0.10 was considered a trend. Explanatory variables were first examined using univariate models before inclusion in the full multivariate model. All predictors with P < 0.2 were maintained for the multivariable model. The risk factors for each of the 10 pathogens were analyzed using multivariable logistic regression (
      • Pardon B.
      • Callens J.
      • Maris J.
      • Allais L.
      • Van Praet W.
      • Deprez P.
      • Ribbens S.
      Pathogen-specific risk factors in acute outbreaks of respiratory disease in calves.
      ). The multivariable model was built with a stepwise approach by backward elimination, gradually excluding nonsignificant variables (
      • Fulton R.W.
      • Blood K.S.
      • Panciera R.J.
      • Payton M.E.
      • Ridpath J.F.
      • Confer A.W.
      • Saliki J.T.
      • Burge L.T.
      • Welsh R.D.
      • Johnson B.J.
      • Reck A.
      Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments.
      ;
      • Dubrovsky S.A.
      • Van Eenennaam A.L.
      • Karle B.M.
      • Rossitto P.V.
      • Lehenbauer T.W.
      • Aly S.S.
      Bovine respiratory disease (BRD) cause-specific and overall mortality in preweaned calves on California dairies: The BRD 10K study.
      ). The Wilcoxon rank sum test was performed to describe the statistical association between BRDC and age. The specific interactions tested included 2-way interaction terms of BSC pneumonia and “other” pneumonia between weaning age and breeds (
      • Fulton R.W.
      • Blood K.S.
      • Panciera R.J.
      • Payton M.E.
      • Ridpath J.F.
      • Confer A.W.
      • Saliki J.T.
      • Burge L.T.
      • Welsh R.D.
      • Johnson B.J.
      • Reck A.
      Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments.
      ;
      • Dubrovsky S.A.
      • Van Eenennaam A.L.
      • Karle B.M.
      • Rossitto P.V.
      • Lehenbauer T.W.
      • Aly S.S.
      Bovine respiratory disease (BRD) cause-specific and overall mortality in preweaned calves on California dairies: The BRD 10K study.
      ). Because the sample sizes were small, percentages were compared using Fisher's exact test. For the final models, pairwise comparisons for categorical predictors were performed using Pearson's chi-squared test.

      RESULTS

      Pathogens Detected in Infected Lungs of Cattle with BRDC

      More than 18 species of pathogens, including 491 strains of bacteria, viruses, or Mycoplasma species, were detected in 160 lung tissue samples of cattle with BRDC. As shown in Table 1, more than 6 species of bacteria were detected, with a positivity rate of 31.77% (156/491). The pathogen positivity rate was the highest for T. pyogenes at 9.37% (46/491), followed by P. multocida (8.35%, 41/491). The positivity rate of Mycoplasma spp. was 38.9% (191/491). The detection rate of M. bovis was 7.74% (38/491) and that of M. dispar was 11.61% (57/491). The 6 viruses were detected with a positivity rate of 29.33% (144/491). The pathogen positivity rate was the highest for BoHV-1 at 13.25% (55/491), and BRSV was detected in 5.50% (27/491) of cases. The pathogenic species of BRDC were shown to be diverse in Chinese cattle herds (Supplemental Table S3).
      Table 1Characteristics of pathogenic infection from 160 bovine respiratory disease complex (BRDC) cases in northeast China between 2016 and 2020
      A total of 491 pathogens were detected by PCR or reverse transcription-PCR from lung tissues of 160 BRDC cases, of which 156 bacterial strains accounted for 31.77%, 191 Mycoplasma strains accounted for 38.9%, and 144 viral strains accounted for 29.33%.
      PathogenNo. of pathogens or casesPathogens/total pathogens, % (n = 491)Cases/total cases, % (n = 160)
      Percentage of cases with single pathogen infection and mixed infection with different types of single pathogens.
      Bacteria15631.77
      Pasteurella multocida418.3525.63
      Trueperella pyogenes469.3728.75
      Mannheimia haemolytica122.447.50
      Histophilus somni224.4813.75
      Escherichia coli173.4610.63
      Streptococcus112.246.88
       Other bacteria71.434.38
      Mycoplasmaspp.19138.9
      M. dispar5711.6135.63
      M. bovis387.7423.75
      M. bovirhinis397.9424.38
      M. alkalescens306.1118.75
      M. arginini40.812.50
       Undetermined species234.6814.38
      Virus14429.33
       Bovine herpesvirus-15511.2034.38
       Bovine viral diarrhea virus244.8915.00
       Bovine parainfluenza virus type-3214.2813.13
       Bovine parainfluenza virus type-561.223.75
       Bovine respiratory syncytial virus275.5016.88
       Bovine coronavirus112.246.88
      Mixed infection14288.75
      Mixed infection of bacterium and other pathogens11873.75
      Mixed infection of virus and other pathogens8050
      Mixed infection of M. bovis and other pathogens3823.75
      1 A total of 491 pathogens were detected by PCR or reverse transcription-PCR from lung tissues of 160 BRDC cases, of which 156 bacterial strains accounted for 31.77%, 191 Mycoplasma strains accounted for 38.9%, and 144 viral strains accounted for 29.33%.
      2 Percentage of cases with single pathogen infection and mixed infection with different types of single pathogens.

      Infection Characteristics of BRDC Cases

      Pathogen Infection Rate

      As shown in Table 1, in bacterial infections among the 160 BRDC cases, T. pyogenes had the highest positivity rate at 28.75% (n = 46), P. multocida accounted for 25.63% (n = 41), H. somni for 13.75% (n = 22), and Mann. haemolytica for 7.5% (n = 12). The positivity rate of M. bovis infection was 23.8% (n = 38). In virus infections, BoHV-1 positivity was the highest at 34.38% (n = 55), followed by BRSV at 16.88% (n = 27), BVDV at 15% (n = 24), BPIV-3 at 13.13% (n = 21), BCoV at 6.88% (n = 11), and BPIV-5 at 3.75% (n = 6).

      Multipathogen Mixed Infection Rate

      The multipathogen mixed infection rate is shown in Table 1. The main form of pathogenic infection was a multipathogen mixed infection, accounting for 88.75% (142/160) of cases. The mixed infection ratio of bacteria and other pathogens was 73.75% (n = 118), followed by that of M. bovis and other pathogens (23.75%, n = 38), and the mixed infection rate of viruses and other pathogens was 50% (n = 80). These results indicated that multipathogen mixed infection was the main cause of death of BRDC cattle.

      Risk of Pathogen Co-Infection

      The risk analysis results for multipathogen co-infection are shown in Table 2. Bovine herpesvirus-1 and H. somni showed a trend toward collaborative infection [P = 0.0032, odds ratio (OR) = 4.14]. In addition, BVDV and M. bovis (P = 0.0334, OR = 2.86), P. multocida (P = 0.0241, OR = 2.88), T. pyogenes (P = 0.0076, OR = 3.44), and Mann. haemolytica (P = 0.0031, OR = 7.22) were associated with co-infection. Moreover, BPIV-3 and M. bovis showed a trend toward collaborative infection (P = 0.0001, OR = 7.41), and BRSV and M. bovis (P = 0.0021, OR = 4.09), P. multocida (P = 0.0004, OR = 5.14), and T. pyogenes (P = 0.0187, OR = 2.87) showed a trend toward collaborative infection. Pasteurella multocida and T. pyogenes showed a trend toward collaborative infection (P = 0.046, OR = 2.2), and M. bovis with T. pyogenes (P = 0.0009, OR = 4.53) and H. somni (P = 0.0152, OR = 3.27) showed a trend toward collaborative infection. The above results indicate that the viruses BVDV, BRSV, and BoHV-1 may be the main risk factors for secondary bacterial infection in BRDC, consistent with previous reports (
      • Shahriar F.M.
      • Clark E.G.
      • Janzen E.
      • West K.
      • Wobeser G.
      Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia.
      ;
      • Pardon B.
      • Callens J.
      • Maris J.
      • Allais L.
      • Van Praet W.
      • Deprez P.
      • Ribbens S.
      Pathogen-specific risk factors in acute outbreaks of respiratory disease in calves.
      ).
      Table 2Risk analysis of the interaction of pathogens in mixed infections based on PCR detection of lung tissues from 160 bovine respiratory disease complex (BRDC) cases
      Dependent variableIndependent variableCategoryCo-infected pathogens in BRDC cases, % (n/m)
      Where n = co-infected pathogens in BRDC cases, and m = number of BRDC cases.
      P-valueOdds ratio95% CI
      Bovine herpesvirus-1Histophilus somniPositive63.64 (14/22)0.00324.141.61–10.63
      Negative29.71 (41/138)
      Bovine viral diarrhea virusMycoplasma bovisPositive26.32 (10/38)0.03342.861.15–7.11
      Negative11.47 (14/122)
      Pasteurella multocidaPositive26.83 (11/41)0.02412.880.56–3.68
      Negative10.92 (13/119)
      Trueperella pyogenesPositive28.26 (13/46)0.00763.441.40–8.42
      Negative9.65 (11/104)
      Mannheimia haemolyticaPositive50 (6/12)0.00317.222.10–24.82
      Negative12.816 (18/148)
      Bovine parainfluenza virus type-3M. bovisPositive34.2 (13/38)0.00017.412.78–19.78
      Negative6.55 (8/122)
      Bovine respiratory syncytial virusM. bovisPositive39.47 (15/38)0.00214.091.71–9.76
      Negative9.84 (12/122)
      P. multocidaPositive36.59 (15/41)0.00045.142.15- 12.3
      Negative10.08 (12/119)
      T. pyogenesPositive28.26 (13/46)0.01872.871.23–6.72
      Negative12.28 (14/114)
      P. multocidaT. pyogenesPositive36.96 (17/46)0.0462.21.04–4.65
      Negative21.05(24/114)
      M. bovisT. pyogenesPositive34.78 (16/46)0.00094.531.93–10.63
      Negative10.53 (12/114)
      H. somniPositive45.45 (10/22)0.01523.271.28–8.35
      Negative20.29 (28/138)
      1 Where n = co-infected pathogens in BRDC cases, and m = number of BRDC cases.

      Risk of Pathogen Infection for BSC Pneumonia

      Statistical analysis of the pathogens detected in the lung tissue of BRDC cases showed that BSC pneumonia cases in BVDV-positive cases accounted for 70.83% (17/24). This was significantly higher than the 36.76% (50/136) cases of BSC pneumonia in BVDV-negative cases (P = 0.0029, OR = 4.18). In M. bovis infection-positive cases, BSC pneumonia cases accounted for 57.89% (22/38), which was significantly higher than the 36.89% (45/122) cases of BSC pneumonia in M. bovis negative cases (P = 0.025, OR = 2.35). The cases of BSC pneumonia in H. somni and T. pyogenes positive cases accounted for 72.73% (16/22) and 82.61% (38/46), respectively, significantly higher than cases of BSC pneumonia in H. somni- and T. pyogenes-negative cases (P < 0.001). The results of the statistical analyses are shown in Table 3, and indicate that lung lesions of BRDC cattle were related to the infected pathogen species.
      Table 3Pathogenic risk factors of 67 cases of bovine suppurative or caseous necrotizing (BSC) pneumonia in 160 bovine respiratory disease complex (BRDC) cases
      Independent variableCategoryBSC pneumonia, % (n/m)
      Where n = number of BSC pneumonia cases and m = number of BRDC cases.
      P-valueOdds ratio95% CI
      Bovine herpesvirus-1Positive49.09 (27/55)0.23751.570.81–3.03
      Negative38.1 (40/105)
      Bovine viral diarrhea virusPositive70.83 (17/24)0.00294.181.62–10.77
      Negative36.76 (50/136)
      Bovine parainfluenza virus type-3Positive33.33 (7/21)0.48050.660.25–1.73
      Negative43.17 (60/139)
      Bovine respiratory syncytial virusPositive25.93 (7/27)0.08670.430.17–1.08
      Negative45.11 (60/133)
      Mycoplasma bovisPositive57.89 (22/38)0.02502.351.12–4.94
      Negative36.89 (45/122)
      Histophilus somniPositive72.73 (16/22)0.00018.172.62–25.52
      Negative36.96 (51/138)
      Pasteurella multocidaPositive51.22 (21/41)0.19921.670.82–3.41
      Negative38.66 (4/119)
      Mannheimia haemolyticaPositive50 (6/12)0.55961.430.44–4.63
      Negative41.23 (61/148)
      Trueperella pyogenesPositive82.61 (38/46)0.000113.925.82–33.28
      Negative25.44 (29/114)
      1 Where n = number of BSC pneumonia cases and m = number of BRDC cases.

      Risk Factors of BRDC

      To explore the risk factors affecting the occurrence of BRDC in northeast China, we performed a cross-sectional epidemiological analysis of the 81 cattle herds submitted for inspection from July 2016 to July 2021. The positive rate of farms (P) was used to assess the association of BRDC with cattle age:
      P=No.offarmswithdiseasedcattleatcorrespondingageTotalno.ofcattlefarms×100%.


      As shown in Figure 2, the positive rate of farms of 0- to 1-mo-old, 1- to 2-mo-old, and 2- to 3-mo-old calves with BRDC was higher than that at other ages. Especially, 1- to 2-mo-old calves had the highest rate (58.02%), which was significantly higher than that of 2- to 3-mo-old calves (P < 0.05). By 8 mo of age, BRDC incidence decreased significantly (P < 0.01) to 8.6% (7/81).
      Figure thumbnail gr2
      Figure 2(a) Statistical heat graph representing significant differences in the positive rate of farms with bovine respiratory disease complex (BRDC) and (b) bar chart representing the positive and negative rate of farms with BRDC in calves of different ages.
      The positivity rate of BRDC in dairy herds was 69.11% (47/68) from 0 to 3 mo of age, which was significantly higher than that of beef herds at 30.88% (21/68; P < 0.001, OR = 5.388). Thus, dairy cattle are more susceptible to BRDC than beef cattle within the first 3 mo of life. However, beef cattle older than 3 mo are more likely to develop BRDC than Holstein dairy cattle (Table 4).
      Table 4Correlation analysis of age and breed (dairy vs. beef breeds) in 160 bovine respiratory disease complex cases
      AgeDairy cattle, % (n1/m1)
      Where n1 = number of dairy cattle; n2 = number of beef cattle; m1 = number of cattle <3 mo; m2 = number of cattle >3 mo.
      Beef cattle, % (n2/m2)
      Where n1 = number of dairy cattle; n2 = number of beef cattle; m1 = number of cattle <3 mo; m2 = number of cattle >3 mo.
      P-valueOdds ratio95% CI
      0–3 mo69.11 (47/68)30.88 (21/68)<0.00015.392.72–10.67
      >3 mo29.35 (27/92)70.65 (65/92)
      1 Where n1 = number of dairy cattle; n2 = number of beef cattle; m1 = number of cattle <3 mo; m2 = number of cattle >3 mo.
      In BRDC cases, the probability of BSC pneumonia occurring in beef cattle was 51.16% (44/86), significantly higher than the probability of BSC pneumonia in dairy cows at 31.08% (23/74) (P = 0.0156, OR = 2.32; Table 5).
      Table 5Correlation analysis between the breeds and bovine suppurative or caseous necrotizing (BSC) pneumonia in 160 bovine respiratory disease complex (BRDC) cases
      VariableBSC pneumonia, % (n/m)
      Where n = number of BSC pneumonia cases; m = number of BRDC cases.
      P-valueOdds ratio95% CI
      Beef cattle51.16 (44/86)0.01562.321.21–4.44
      Dairy cattle31.08 (23/74)
      1 Where n = number of BSC pneumonia cases; m = number of BRDC cases.

      Molecular Epidemiological Survey of Pathogens from BRDC Cases

      Mycoplasma spp

      To determine the species of Mycoplasma in BRDC cases, the 103 Mycoplasma-positive samples were classified by PCR using specific primers for the 6 bovine Mycoplasma spp. that infect the bovine respiratory tract. As shown in Table 1, 191 Mycoplasma strains were detected; M. dispar had the highest detection rate of 29.84% (57/191), followed by M. bovirhinis (20.42%, 39/191). In contrast, the detection rate of traditionally highly pathogenic M. bovis was only 19.9% (38/191). In addition, M. alkalescens was positive in 15.71% (30/191) and M. arginini was positive in 2.09% (4/191) of cases. Mycoplasma mycoides ssp. mycoides was not detected in any of the samples. In addition, the Mycoplasma-positive samples that could not be typed accounted for 12.04% (23/191) of cases. We analyzed 80 cases with known Mycoplasma species and found that co-infection with Mycoplasma species was prevalent, accounting for 61.25% (n = 49). Single Mycoplasma spp. infections accounted for 38.75% (n = 31), followed by infections with 2 types at 25.5%, 3 at 10.8%, 4 at 7.77%, and 5 at 2.91% (Figure 3).
      Figure thumbnail gr3
      Figure 3Characteristics of individual infection or co-infection of Mycoplasma species in 80 bovine respiratory disease complex (BRDC) cases in which specific Mycoplasma species were detected.

      P. multocida

      Pasteurella multocida has 5 capsular serotypes. To determine the serotype currently prevalent in BRDC cases of cattle herds in China, 36 P. multocida strains were isolated from the 41 PCR-positive lung tissue samples of cattle with BRDC from 26 cattle farms in northeast China. Two capsular serotypes of P. multocida were detected. Serotype A strains were positive for 91.67% (33/36) and serotype D strains were positive for 8.33% (3/36) of cases. No strains of serotype B, E, or F were detected. Three serotype D cases were isolated from the same cattle herd in July 2019 and September 2020. This is the first report of the isolation of serotype D strains from cattle.

      Isolation, Identification, and Genetic Evolution Analysis of BoHV-1

      Among the 55 BoHV-1-positive samples inoculated on MDBK cells, 42 samples showed BoHV-1 typical CPE (grape string or aggregation) after 1 to 3 passages, and the isolated BoHV-1 was determined by PCR amplification of the 339-bp gB gene fragment. The 13 BoHV-1 isolates with regional representation were selected to amplify the gC gene for genetic evolution analysis. According to the phylogenetic analysis of a 451-bp gC gene fragment, all 13 BoHV-1 isolates were classified into the BoHV-1.2 gene subtype but located in 2 phylogenetic tree branches with a long genetic distance (Figure 4). One of the 2 branched BoHV-1 strains accounted for 46.15% (6/13) of the BoHV-1.2b gene subtype. The other branch BoHV-1 strains accounted for 53.85% (7/13), which was different from the traditional BoHV-1.2a and -1.2b gene subtype, and was named the BoHV-1.2c gene subtype, as described recently by our group (
      • Zhou Y.
      • Li X.
      • Ren Y.
      • Hou X.
      • Liu Y.
      • Wei S.
      • Dai G.
      • Meng Y.
      • Hu L.
      • Liu Z.
      • Jia W.
      • Zhu Z.
      • Wu R.
      Phylogenetic analysis and characterization of bovine herpesvirus-1 in cattle of China, 2016–2019.
      ).
      Figure thumbnail gr4
      Figure 4Phylogenetic analysis of bovine herpesvirus-1 (BoHV-1) based on 451 nucleotides of partial gC gene. The tree includes 13 BoHV-1 isolates (•) and 8 reference strain sequences from GenBank (Supplemental Table S3; https://doi.org/10.6084/m9.figshare.21341556 ).

      Isolation, Identification, and Genetic Evolution Analysis of BVDV

      The MDBK cells were inoculated with 24 BVDV-positive lung samples identified by RT-PCR. After the third blind passage, 15 BVDV isolates were identified by RT-PCR using the 5′-UTR specific primer; 12 BVDV isolates were of the cytopathic (CP) biotype (cell lengthening, stringing, intercellular space enlargement), and 3 BVDV isolates were noncytopathic (NCP) biotypes. The 288-bp 5′-UTR products of the 15 BVDV isolates were sequenced, and a 250-bp gene sequence was used for genetic evolution analysis. As shown in Figure 5, 2 BVDV-1 subtypes were detected: BVDV-1b was detected in 53.3% (8/15) and BVDV-1d in 46.7% (7/15) of cases.
      Figure thumbnail gr5
      Figure 5The phylogenetic tree was created using the 5′-UTR (untranslated region) nucleotide sequences from 15 bovine viral diarrhea virus (BVDV) isolates (•) from lung samples of bovine respiratory disease complex (BRDC) cases and 23 BVDV reference strains retrieved from GenBank (Supplemental Table S4; https://doi.org/10.6084/m9.figshare.21341556 ).

      Isolation, Identification, and Genetic Evolution Analysis of BPIV-3

      Among the 21 BPIV-3-positive lung samples used to inoculate the MDBK cells, 17 showed BPIV-3 typical CPE (cell swelling, rounding, shedding, and cell fusion) after 1 to 3 passages. The 330-bp BPIV-3 M gene fragment from the 17 BPIV-3 isolates was amplified by RT-PCR and sequenced for genetic evolution analysis. The results revealed 2 genotypes in these BPIV-3 isolates: BPIV-3a was found in 29.41% (5/17) and BPIV-3c was observed in 70.59% (12/17) of cases (Figure 6). Another genotype, BPIV-3b, was not detected in this study.
      Figure thumbnail gr6
      Figure 6Phylogenetic analysis of bovine parainfluenza virus type-3 (BPIV-3) based on 330 nucleotides of partial M gene. The tree included 17 BPIV-3 isolates (•) and 3 reference strain sequences from GenBank (Supplemental Table S4; https://doi.org/10.6084/m9.figshare.21341556 ).

      Genetic Evolution Analysis of BRSV

      In this study, 2 lung tissue samples with typical BRSV infection lesions from 2 cattle herds with high mortality caused by BRDC in Qiqihar City (dairy cattle herd) and Anda City (beef cattle herd) were identified as BRSV positive by RT-PCR and used for BRSV G gene sequencing. The 848-bp gene fragment of the BRSV G gene was obtained by reverse transcription and semi-nested PCR (
      • Valentova V.
      • Antonis A.F.
      • Kovarcik K.
      Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates.
      ), and the 750-bp G gene fragments from the 2 samples were sequenced for genetic evolution analysis. The similarity of the G gene sequence was 98.8% between the 2 BRSV strains and was 85.3 to 89.7% among the 2 strains and the gene types I–VIII of BRSV strains from GenBank (Table 6). In the phylogenetic analysis, the 2 BRSV strains were located on the same branch, independent of the previously reported genotypes (Figure 7) and were tentatively named genotype IX. These results indicated that BRSV shows clear genetic variation in northeast China.
      Table 6Percentage of similarity generated by pairwise comparison of G gene sequences of bovine respiratory syncytial virus (BRSV) isolates and different BRSV genotypes
      Virus strainGenBank accession no.Classification
      References, I–VI: Krešić et al, 2018 and Valarcher et al, 2000; VII–IX: Krešić et al, 2018.
      Nucleotide sequence similarity
      BRSV isolates ANDA and QQHER from Heilongjiang Province, China.
      ANDAQQHER
      4642Y08718I85.785.7
      BovXU57823I85.585.5
      8307027BRU92098II87.587.5
      FV160AF188578II87.587.5
      BRSATTGLYFL08415III89.789.7
      391.2M58307III87.587.3
      SNOOKY08719IV88.188.1
      DorsetBRU24715IV87.587.5
      88PAF188604V86.986.9
      58PAF188603V87.187.1
      75PAF188587VI87.787.7
      K1AF188585VI87.387.3
      B29/12/CROKY680321VII85.385.7
      B26/12/CROKY680320VII85.385.7
      B60279/2/14/CROKY680330VIII87.587.5
      B10152/2/16/CROKY680334VIII86.386.3
      A2M11486HRSV54.454.6
      ANDAOM372493IX10098.8
      1 References, I–VI:
      • Krešić N.
      • Bedeković T.
      • Brnić D.
      • Šimić I.
      • Lojkić I.
      • Turk N.
      Genetic analysis of bovine respiratory syncytial virus in Croatia.
      and
      • Valarcher J.F.
      • Schelcher F.
      • Bourhy H.
      Evolution of bovine respiratory syncytial virus.
      ; VII–IX:
      • Krešić N.
      • Bedeković T.
      • Brnić D.
      • Šimić I.
      • Lojkić I.
      • Turk N.
      Genetic analysis of bovine respiratory syncytial virus in Croatia.
      .
      2 BRSV isolates ANDA and QQHER from Heilongjiang Province, China.
      Figure thumbnail gr7
      Figure 7Phylogenetic analysis of bovine respiratory syncytial virus (BRSV) based on 750 nucleotides of partial G gene. The tree included 2 BRSV strains (•) and 17 reference strain sequences from GenBank (Supplemental Table S4; https://doi.org/10.6084/m9.figshare.21341556 ).

      DISCUSSION

      The etiology of BRDC is complicated and involves many types of pathogens infecting the bovine respiratory tract, including viruses, bacteria, and Mycoplasma spp. (
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ;
      • Alexander T.W.
      • Timsit E.
      • Amat S.
      The role of the bovine respiratory bacterial microbiota in health and disease.
      ). Pathological necropsy and pathogen detection in deaths from acute or chronic BRDC can reflect BRDC pathogen infection characteristics. Few epidemiological studies are based on detecting pathogens from lung samples because of the difficulty in collecting lung tissue samples from dead cattle. Given this, we sampled 160 lungs from cattle that died from BRDC collected from 2016 to 2021 as pathogen detection samples. Our results revealed that 3 times more pathogens (491 strains) than the number of cases (160 cases) were detected, which shows that multi-pathogen mixed infection [88.75% (142/160) of cases] was the leading cause of death in BRDC. These findings suggested that the prevention and treatment of BRDC should include comprehensive measures against bacteria, viruses, and Mycoplasma to achieve the desired effect, and that it is important to develop a combined vaccine to prevent BRDC. Considering that the 160 lung samples in this study were all from BRDC cases resulting in natural deaths (animals that were in the late stage of the disease and had a long disease course), some of the primary viral pathogens infected in the early stage would have disappeared, whereas opportunistic pathogens proliferated in large numbers. Thus, the type and quantity of viral pathogens detected in this study might be lower than that during actual infection, whereas the detection rate of opportunistic pathogens exceeds the infection rate in the early stage of the disease. In addition, these cases were treated with antibiotics, which could have resulted in the suppression of certain antibiotic-susceptible bacteria or mycoplasma pathogens. Because the main pathogenic bacteria in BRDC are aerobic bacteria, M. bovis, T. pyogenes, and H. somni do not need to be cultured under strict anaerobic conditions, so strict anaerobic culture was not performed in this study. There are likely more anaerobes present than the most common pathogens of BRDC, and the lack of strict anaerobic culture was a weakness of this study.
      The primary infectious pathogens in the BRDC epidemic were BoHV-1, BVDV, BRSV, and BPIV-3. The positivity rate of BoHV-1 in the lungs of BRDC cases was the highest at 34.38% (Table 2), which is higher than the positivity rate (24.83%) of nasal swabs from animals with BRDC living in Inner Mongolia, China, which may be related to differences in the tested samples from different sources (
      • Guo T.
      • Zhang J.
      • Chen X.
      • Wei X.
      • Wu C.
      • Cui Q.
      • Hao Y.
      Investigation of viral pathogens in cattle with bovine respiratory disease complex in Inner Mongolia, China.
      ). It is important to note that a new subgenotype of BoHV-1, BoHV-1.2c, was highly endemic to northeast China, accounting for 53.85% (7/13) of cases. In China, BVDV strains in cattle herds are highly diverse, including 2 genotypes and many subgenotypes (BVDV-1a, b, c, d, m, p, q, u, and BVDV-2a;
      • Hou P.
      • Zhao G.
      • Wang H.
      • He H.
      Prevalence of bovine viral diarrhea virus in dairy cattle herds in eastern China.
      ). However, BVDV-1b (50%, 8/16) and BVDV-1d (50%, 8/16) were the dominant epidemic strains in the BRDC cases in northeast China. The BVDV-1b subgenotype is epidemic in almost all countries where sequence data exist; BVDV-1d is prevalent in many countries, including China, Germany, Italy, Poland, Turkey, Slovenia, Austria, and Japan (
      • Yeşilbağ K.
      • Alpay G.
      • Becher P.
      Variability and global distribution of subgenotypes of bovine viral diarrhea virus.
      ). Thus, we speculate that BVDV-1b and 1d are also the main genotypes currently prevalent in Chinese cattle herds. Bovine parainfluenza virus type-3 has 3 subgenotypes (a, b, and c) and only BPIV-3a and BPIV-3c were detected in northeast China, of which BPIV-3c was the dominant endemic strain with a positivity rate of 70.59% (12/17). The well-understood sensitivity of BRSV has resulted in inefficient virus isolation from clinical samples (
      • Valentova V.
      • Antonis A.F.
      • Kovarcik K.
      Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates.
      ). We attempted to isolate BRSV from clinical samples but were unsuccessful. Thus, 2 representative BRSV RT-PCR–positive samples were selected for G gene sequencing for molecular epidemiological investigation. According to the genetic evolution analysis of the BRSV G gene as shown in Figure 7, the 2 BRSV strains detected in this study could not be classified to the one of the gene subgroups I to VIII previously described (
      • Valentova V.
      • Antonis A.F.
      • Kovarcik K.
      Restriction enzyme analysis of RT-PCR amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates.
      ;
      • Krešić N.
      • Bedeković T.
      • Brnić D.
      • Šimić I.
      • Lojkić I.
      • Turk N.
      Genetic analysis of bovine respiratory syncytial virus in Croatia.
      ). Therefore, they were temporarily designated as belonging to novel gene subgroup IX. This is important for further research on the genetic evolution and pathogenic mechanisms of BRSV. As shown in Supplemental Figure S4g ( https://doi.org/10.6084/m9.figshare.21341544 ), the BRSV ANDA strain, M. bovis, M. dispar, and P. multocida serotype A were simultaneously detected in lung tissue of cattle with BRDC, which might be due to the immunosuppressive effect caused by BRSV increasing the risk of infection by other pathogens (
      • Gershwin L.J.
      Bovine respiratory syncytial virus infection: Immunopathogenic mechanisms.
      ). The lungs showed clear emphysema, fleshy lesions, suppurative foci, caseous necrosis foci, and fibrinous pneumonia pathological lesions. Lung emphysema was also seen in bovine lungs infected with the BRSV QQHER strain and other BRSV strains in previous reports (
      • Gershwin L.J.
      Bovine respiratory syncytial virus infection: Immunopathogenic mechanisms.
      ). Therefore, lung emphysema can be a characteristic of severe cases of BRSV. This study explored the association among co-infected pathogens in the lungs of BRDC cases. To meet the statistical requirements, co-infection by 2 pathogens was not analyzed when <6 cases were identified. Co-infecting pathogens without significant differences are not listed in Table 2. As shown in Table 2, the viruses BVDV, BRSV, and BoHV-1 were the main risk factors for secondary bacterial infection in BRDC. This might be related to the disturbance of the host's immune system, including breakdown of the upper respiratory tract mucosa, imbalance of flora, and immunosuppression caused by persistent viral infection through immune evasion (
      • Gershwin L.J.
      Bovine respiratory syncytial virus infection: Immunopathogenic mechanisms.
      ;
      • Srikumaran S.
      • Kelling C.L.
      • Ambagala A.
      Immune evasion by pathogens of bovine respiratory disease complex.
      ;
      • Jones C.
      Bovine herpesvirus 1 counteracts immune responses and immune-surveillance to enhance pathogenesis and virus transmission.
      ). Cattle infected with BVDV appeared predisposed to bacterial pneumonia, especially to co-infections with P. multocida (P = 0.02, OR = 2.88), Mann. haemolytica (P < 0.01, OR = 7.22), T. pyogenes (P < 0.01, OR = 3.44), and M. bovis (P = 0.03, OR = 2.86), which is consistent with previous reports (
      • Caswell J.L.
      • Bateman K.G.
      • Cai H.Y.
      • Castillo-Alcala F.
      Mycoplasma bovis in respiratory disease of feedlot cattle.
      ).
      Autopsies of cattle with BRDC showed that the lung lesions were complex and diverse; BSC pneumonia cases accounted for 41.88% (67/160; Table 3) of cases. The lung lesions were characterized by caseous necrosis caused by M. bovis infection, purulent pneumonia caused by T. pyogenes infection, and fibrinous and purulent pneumonia caused by P. multocida, Mann. haemolytica, or H. somni (
      • Caswell J.L.
      • Bateman K.G.
      • Cai H.Y.
      • Castillo-Alcala F.
      Mycoplasma bovis in respiratory disease of feedlot cattle.
      ;
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ;
      • Bassel L.L.
      • Caswell J.L.
      Bovine neutrophils in health and disease.
      ).
      Because the above-mentioned pathogens are often present as co-infections resulting in atypical lung lesions, we referred to this type of pneumonia as “BSC pneumonia.” The BSC pneumonia is a characteristic type of chronic pneumonia associated with BRDC and is mostly caused by bacteria that infect the lungs and induce leukocytes to migrate to the lungs. In contrast, neutrophils clear pathogens through oxidative bursts, phagocytosis, or neutrophil extracellular traps (NETs;
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • Weinrauch Y.
      • Zychlinsky A.
      Neutrophil extracellular traps kill bacteria.
      ;
      • Bassel L.L.
      • Caswell J.L.
      Bovine neutrophils in health and disease.
      ). Neutrophils have protective bactericidal effects but also induce tissue damage by releasing proteases, oxygen radicals, or cytotoxic effects (
      • Bassel L.L.
      • Caswell J.L.
      Bovine neutrophils in health and disease.
      ).
      An important finding of the current study was the incidence that BVDV, M. bovis, H. somni, and T. pyogenes infection was significantly higher in BSC pneumonia than in other types of pneumonia (Table 3), suggesting that M. bovis, H. somni, and T. pyogenes might be secondary infections of BVDV in BSC pneumonia. Primary BVDV infection causes neutropenia and is a risk factor for secondary infection of bacterial pneumonia. This is also common in cattle with caseonecrotic bronchopneumonia (
      • Shahriar F.M.
      • Clark E.G.
      • Janzen E.
      • West K.
      • Wobeser G.
      Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia.
      ;
      • Gagea M.I.
      • Bateman K.G.
      • Shanahan R.A.
      • van Dreumel T.
      • McEwen B.J.
      • Carman S.
      • Archambault M.
      • Caswell J.L.
      Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves.
      ;
      • Keller S.L.
      • Jefferson B.J.
      • Jacobs R.M.
      • Wood R.D.
      Effects of noncytopathic type 2 bovine viral diarrhea virus on the proliferation of bone marrow progenitor cells.
      ). Bovine viral diarrhea virus can cause leukopenia and lymphopenia in the early stages of infection and cause a significant decrease in phagocytosis and killing of neutrophils within 2 wk after infection (
      • Gånheim C.
      • Johannisson A.
      • Ohagen P.
      • Persson Waller K.
      Changes in peripheral blood leucocyte counts and subpopulations after experimental infection with BVDV and/or Mannheimia haemolytica..
      ). Moreover, cytopathic or noncytopathic BVDV-infected neutrophils lead directly to a decrease in the expression of CD18 and CD62L, thereby inhibiting neutrophils from exuding blood vessels and migrating to tissues (

      Thakur, N., L. Braun, and C. C. Chase. 2014. Effect of bovine viral diarrhea virus (BVDV) infection on neutrophil survival and surface marker expression. Abstract 71P. Proc. 95th Ann. Mtg. Conf. Research Workers in Animal Diseases, Chicago, IL, Dec. 7–9, 2014.

      ). It also leads to increased expression of CD14, which mainly recognizes LPS from the cell wall of gram-negative bacteria (

      Thakur, N., L. Braun, and C. C. Chase. 2014. Effect of bovine viral diarrhea virus (BVDV) infection on neutrophil survival and surface marker expression. Abstract 71P. Proc. 95th Ann. Mtg. Conf. Research Workers in Animal Diseases, Chicago, IL, Dec. 7–9, 2014.

      ). Lipopolysaccharide binds to the CD14 receptor of neutrophils, leading to increased production of pro-inflammatory factors tumor necrosis factor-α and IL-1 (
      • Sohn E.J.
      • Paape M.J.
      • Bannerman D.D.
      • Connor E.E.
      • Fetterer R.H.
      • Peters R.R.
      Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8.
      ;
      • Molina V.
      • Risalde M.A.
      • Sánchez-Cordón P.J.
      • Romero-Palomo F.
      • Pedrera M.
      • Garfia B.
      • Gómez-Villamandos J.C.
      Cell-mediated immune response during experimental acute infection with bovine viral diarrhoea virus: Evaluation of blood parameters.
      ). Therefore, we speculate that secondary bacterial infection may be due to BVDV infection causing early leukopenia in the body, inhibiting the migration of neutrophils to lung tissue with bacterial infection, reducing the killing function of neutrophils, and promoting the pro-inflammatory reaction caused by gram-negative bacteria.
      In this study, P. multocida and Mann. haemolytica showed no significant difference in infection rates between BSC and “other” pneumonia. This may be because antibiotics are widely used to treat BRDC and the bacteria may be suppressed (
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ). Worldwide, P. multocida serogroup A isolates are a major cause of BRDC (
      • Dabo S.M.
      • Taylor J.
      • Confer A.
      Pasteurella multocida and bovine respiratory disease.
      ;
      • Ma W.G.
      • Jiang Z.G.
      • Yu L.
      Molecular epidemiological investigation of bovine capsular serotype A pasteurellosis.
      ). In this study, 2 P. multocida serotypes were detected; serotype A was detected in 91.67% (33/36), and serotype D in 8.33% (n = 3/36) of cases. Serotype D is associated with atrophic rhinitis and pneumonia in pigs and with avian cholera. In this study, serotype D was first detected in cattle with BRDC. The 3 serotype D strains were isolated from the same Holstein herd in July 2019 and September 2020. Two serotype D strains were derived from the calves that died from BRDC aged 30 to 60 d and exhibited fibrinous purulent pneumonia. The other strain was isolated from a cow that dies suddenly at 20 d postpartum and exhibited fibrinous necrosis bronchial pneumonia. Thus, P. multocida serotype D can infect both calves and adult cattle. It has become a prevalent strain in a bovine herd in northeast China and is a possible pathogen of fibrinous purulent pneumonia and fibrinous necrosis bronchial pneumonia.
      Two forms of lung lesions were observed in BRDC cases when T. pyogenes was isolated. One was pleural effusion, where the surface is covered with fibrin, the alveolar collapses and shrinks, and the bronchioles or alveoli are fused with purulent foci with pus inside (Supplemental Figure S1a). The other was where pustules of different sizes were apparent in most areas of the cardiac lobe and apical lobe of the lung with connective tissue cysts outside, and the contents became a viscous paste or liquid (Supplemental Figure S3e). However, pleural effusion was not observed. All T. pyogenes infection cases were multipathogen infections. Trueperella pyogenes is regarded as a secondary infectious bacterium in BRDC, and its pathogenic effects have been neglected (
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ). However, in this study, the bacterial species with the highest detection rate was T. pyogenes, which reached 28.75% (46/160).
      • Fulton R.W.
      Bovine respiratory disease research (1983–2009).
      also showed that T. pyogenes had the highest detection rate (35.0%) among lung tissues of cattle with BRDC. Pneumonia caused by T. pyogenes has rarely been reported in animals such as pigs and poultry (
      • Rzewuska M.
      • Kwiecień E.
      • Chrobak-Chmiel D.
      • Kizerwetter-Świda M.
      • Stefańska I.
      • Gieryńska M.
      Pathogenicity and virulence of Trueperella pyogenes: A review.
      ). Trueperella pyogenes isolated from lung tissues of BRDC cases successfully infected goats and created a purulent pneumonia model, suggesting that the pathogenic role of T. pyogenes in BRDC is of concern (
      • Sun Y.J.
      • Li L.
      • Shi Z.Q.
      • Ding M.Y.
      • Zhou Y.
      • Che Y.F.
      • Zhao W.P.
      • Liu L.S.
      • Yan C.L.
      • Fan C.L.
      Pathological changes of goats infected with Arcanobacterium pyogenes from bovine.
      ).
      Mycoplasma bovis is most commonly considered the cause of chronic caseonecrotic bronchopneumonia with or without arthritis (
      • Caswell J.L.
      • Hewson J.
      • Slavić D.
      • DeLay J.
      • Bateman K.
      Laboratory and postmortem diagnosis of bovine respiratory disease.
      ). In addition, M. bovis is increasingly recognized as a primary pathogen, although this remains controversial (
      • Calcutt M.J.
      • Lysnyansky I.
      • Sachse K.
      • Fox L.K.
      • Nicholas R.A.J.
      • Ayling R.D.
      Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements.
      ). However, the economic importance of M. bovis cannot be accurately measured at present because the clinical signs caused by M. bovis are not specific, and some forms of lung lesions caused by M. bovis cannot be easily distinguished from those caused by other bacteria (
      • Caswell J.L.
      • Hewson J.
      • Slavić D.
      • DeLay J.
      • Bateman K.
      Laboratory and postmortem diagnosis of bovine respiratory disease.
      ). Our results showed that all 38 cases of M. bovis infection were co-infected with other pathogens (Table 1), confirming that the co-infection of M. bovis with other respiratory pathogens is a common occurrence (
      • Shahriar F.M.
      • Clark E.G.
      • Janzen E.
      • West K.
      • Wobeser G.
      Coinfection with bovine viral diarrhea virus and Mycoplasma bovis in feedlot cattle with chronic pneumonia.
      ;
      • Gagea M.I.
      • Bateman K.G.
      • Shanahan R.A.
      • van Dreumel T.
      • McEwen B.J.
      • Carman S.
      • Archambault M.
      • Caswell J.L.
      Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves.
      ). Moreover, primary pathogens such as BoHV-1, BVDV, BPIV-3, or BRSV were detected in all 38 cases of M. bovis infection, and T. pyogenes, H. somni, P. multocida, M. dispar, or E. coli were detected in 94.7% (36/38) of M. bovis infection cases. Based on the above, we presumed that M. bovis is a secondary pathogen in Chinese BRDC that can promote bacterial infection of the bovine respiratory tract. We also found that co-infection among members of Mycoplasma spp. is very common. Overall, 25.5% (n = 26) of cases had 2 types of mycoplasmas, and 2.91% (n = 3) of cases had co-infection with 5 types of mycoplasmas (Figure 3). The mycoplasma with the highest detection rate in BRDC cases in this study was M. dispar (35.63%, n = 57), not M. bovis (23.75%, n = 38). In addition, in a few typical caseonecrotic lesions of the lungs, either M. dispar alone was detected or M. bovis was detected together with M. dispar. In our investigation, M. dispar was regularly isolated from bovine pneumonic lungs, and its presence was associated with mild infection. In a previous study, M. dispar was one of the most important causes of respiratory disease in cattle (
      • Tortorelli G.
      • Carrillo Gaeta N.
      Evaluation of mollicutes microorganisms in respiratory disease of cattle and their relationship to clinical signs.
      ). Many studies have indicated that M. dispar is present in 50% of examined bovine herds and that it coexists with other bacterial agents, such as P. multocida, T. pyogenes, and Mann. haemolytica (
      • Bednarek D.
      • Szymańska-Czerwińska M.
      • Dudek K.
      Bovine respiratory syndrome (BRD) etiopathogenesis, diagnosis and control.
      ). Our study showed that M. dispar is an important pathogen in BRDC in Chinese herds and may play a role in the pathogenicity of pathogens associated with BRDC in a form similar to that of M. bovis. Mycoplasma dispar and M. bovirhinis are the most abundant species in the bovine nasopharynx, accounting for 53% of the total bacterial population (
      • Timsit E.
      • Holman D.B.
      • Hallewell J.
      • Alexander T.W.
      The nasopharyngeal microbiota in feedlot cattle and its role in respiratory health.
      ). Although M. bovirhinis is one of the most commonly occurring species in bovine respiratory diseases (
      • Ayling R.D.
      • Bashiruddin S.E.
      • Nicholas R.A.
      Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000.
      ), it is not considered a primary pathogen because it is frequently isolated from healthy or asymptomatic animals and it might be part of the natural bacterial flora. Mycoplasma hyorhinis usually infects swine, leading to respiratory tract disease and inflammation of the chest and joints (
      • Kobisch M.
      • Friis N.F.
      Swine mycoplasmoses.
      ). In addition, accumulating evidence suggests that M. hyorhinis infection in humans results in clinical outcomes (
      • Huang S.
      • Li J.Y.
      • Wu J.
      • Meng L.
      • Shou C.C.
      Mycoplasma infections and different human carcinomas.
      ). The positivity rate of M. bovirhinis was 24.38% (n = 39) in lung tissue of dead cattle with BRDC, suggesting that it may also be an important pathogen of BRDC (
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ). Undetermined mycoplasma species, except for M. mycoides ssp. mycoides SC, M. bovis, M. dispar, M. bovirhinis, M. alkalensis, and M. arginini, were observed in 14.38% (n = 23) of cases this study, showing that there are unidentified mycoplasma species in the lungs of cattle with BRDC. To formulate an effective method to control Mycoplasma infection, it is important to conduct an in-depth study of the pathogenicity of different mycoplasma species in BRDC. The gross lesions of lung tissue are similar between BSC pneumonia and bovine tuberculosis. Mycobacterium bovis was not detected in BSC pneumonia cases by histology, Neelsen staining, or PCR methods in a previous study (
      • Guo T.
      • Qi X.
      • Zhang L.
      • Zhao X.Y.
      • Jia X.J.
      • Xu J.Z.
      • Fan C.L.
      Pathological study of bovine disease of Arcanobacterium pyogenes.
      ;
      • Yao X.R.
      • Yuan J.B.
      • Wang M.M.
      • Lv C.J.
      • Huang B.Y.
      • Ren S.
      • Sun X.R.
      • Sun S.H.
      • Fan C.L.
      Expression of caspase-8, caspase-9 and caspase-3 in bovine lungs with Arcanobacterium pyogenes.
      ; ). However, M. bovis was not tested in the current study, which is a weakness in the study.
      Risk factors for BRDC have been demonstrated in groups comprising animals from multiple sources, including host genetics, mode of delivery, diet and the microbiota of the mother, environmental housing, weaning, feeding type, transportation, commuting, antibiotic treatment, vaccination, and pathogen exposure (
      • Zeineldin M.
      • Lowe J.
      • Aldridge B.
      Contribution of the mucosal microbiota to bovine respiratory health.
      ). We found that the incidence of BRDC in calves ≤3 mo was significantly higher than that in older calves, and the highest BRDC positivity rate was 58%, between 1 and 2 mo of age. This is consistent with the results of a BRDC study of American dairy calves (
      • USDA
      ;
      • Dubrovsky S.A.
      • Van Eenennaam A.L.
      • Karle B.M.
      • Rossitto P.V.
      • Lehenbauer T.W.
      • Aly S.S.
      Bovine respiratory disease (BRD) cause-specific and overall mortality in preweaned calves on California dairies: The BRD 10K study.
      ). Under the current feeding model in China, beef calves rely on cows to feed them until they are weaned at 3 mo; dairy calves are fed milk replacement products and generally weaned at 2 mo. Importantly, weaning stress can also cause BRDC development. Therefore, the reasons described above may cause the incidence rate around 3 mo of age to be significantly higher than that at other ages.
      Breed was identified as an important risk factor for BRDC. Previous studies have provided evidence of the association between breed and BRDC. A reasonable biological approach has been proposed, as genetic susceptibility has been shown to vary between breeds (
      • Snowder G.D.
      • Van Vleck L.D.
      • Cundiff L.V.
      • Bennett G.L.
      Bovine respiratory disease in feedlot cattle: environmental, genetic, and economic factors.
      ;
      • Neibergs H.
      • Zanella R.
      • Casas E.
      • Snowder G.D.
      • Wenz J.
      • Neibergs J.S.
      • Moore D.
      Loci on Bos taurus chromosome 2 and Bos taurus chromosome 26 are linked with bovine respiratory disease and associated with persistent infection of bovine viral diarrhea virus.
      ). Table 4 shows that dairy cows from 0 to 3 mo were more susceptible to BRDC than beef cattle of the same age, whereas beef cattle >3 mo were more susceptible to BRDC than dairy cows of the same age (P < 0.0001, OR = 5.39). In addition, the incidence of dairy cattle suffering from BSC pneumonia was significantly lower than that of beef cattle (P = 0.0156, OR = 2.32). This may be because beef cattle have to be transferred to the fattening field after weaning and experience more severe weaning, transportation, and mixed-group stress than dairy cows. As the age of the calf increases, its immune system matures, and its resistance to pathogens increases, leading to chronic BRDC cases characterized by BSC pneumonia. These observations showed a correlation between the breed and BRDC, most likely due to differences in feeding and management methods in different cattle breeds.
      Bovine respiratory disease complex is a multifactorial syndrome involving multiple pathogens, and it is affected by multiple nonpathogenic factors, such as the host, surrounding environment, and management practices (
      • Buckham Sporer K.R.
      • Weber P.S.
      • Burton J.L.
      • Earley B.
      • Crowe M.A.
      Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress.
      ;
      • McMullen C.
      • Orsel K.
      • Alexander T.W.
      • van der Meer F.
      • Plastow G.
      • Timsit E.
      Comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease.
      ). When cattle encounter different stressors or are infected with bovine respiratory viruses (BoHV-1, BVDV, BRSV, and BPIV-3), their defense ability is weakened. This leads to nasopharyngeal dysbiosis causing abnormal proliferation of pathogenic commensal bacteria, such as Mann. haemolytica, H. somni, P. multocida, T. pyogenes, M. bovis, M. dispar, Ureaplasma diversum, and M. bovirhinis that colonize the upper respiratory tract mucosa and invade the lungs via inhalation (
      • Fulton R.W.
      • Blood K.S.
      • Panciera R.J.
      • Payton M.E.
      • Ridpath J.F.
      • Confer A.W.
      • Saliki J.T.
      • Burge L.T.
      • Welsh R.D.
      • Johnson B.J.
      • Reck A.
      Lung pathology and infectious agents in fatal feedlot pneumonias and relationship with mortality, disease onset, and treatments.
      ;
      • Griffin D.
      • Chengappa M.M.
      • Kuszak J.
      • McVey D.S.
      Bacterial pathogens of the bovine respiratory disease complex.
      ;
      • Timsit E.
      • McMullen C.
      • Amat S.
      • Alexander T.W.
      Respiratory bacterial microbiota in cattle: From development to modulation to enhance respiratory health.
      ).

      CONCLUSIONS

      In the cattle herds of Northeast China, mixed infection with more than 2 pathogens is a clear feature of BRDC; the common pathogens include BoHV-1, T. pyogenes, P. multocida, and M. bovis; BoHV-1.2c, BVDV-1b and 1d, BPIV-3c, BRSV gene subgroup Ⅸ, and serotype A of P. multocida are the popular subgenotypes. Co-infection with multiple mycoplasmas or between mycoplasmas and bacteria or viruses is common in cattle with BRDC. In the case of BSC pneumonia, the main pathogens are BVDV-1, M. bovis, T. pyogenes, and H. somni. Beef cattle are more susceptible to BSC pneumonia than dairy cattle. These data have important guiding significance for the development of multivalent vaccines for the prevention and control of BRDC.

      ACKNOWLEDGMENTS

      This work was supported by the Natural Science Foundation Project of Heilongjiang Province (grant numbers LH2020C083), Heilongjiang Provincial Postdoctoral Research Start-up Foundation (grant numbers LBH-Z18258), Heilongjiang Bayi Agricultural University Startup Program (grant numbers XDB201820), and Heilongjiang University Students Innovation and Entrepreneurship Training Project (grant number 202110223011), provided by the Natural Sciences Foundation Committee and Department of Education of Heilongjiang Province. The authors thank Yu Li, a researcher at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, for his comments during the writing of the manuscript; Zhu Huan (liberal arts college of Heilongjiang Bayi Agricultural University) for his data analysis during the writing of the manuscript; and the veterinarians, ranch managers, and breeders involved in this study who helped collect the samples and data. The authors have not stated any conflicts of interest.

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