Detection of Mycoplasmopsis ( Mycoplasma ) bovis in formalin-fixed,paraffin-embedded bovine lung sections by immunohistochemistry and in situ hybridization

Abstract

The bovine respiratory disease complex (BRDC) is a multifactorial disease of economic importance in cattle involving viral and bacterial agents and several environmental and host-associated predisposing factors. Mycoplasmopsis (Mycoplasma) bovis is frequently detected in BRDC cases, but the role of this bacterium in the pathogenesis of BRDC is not completely understood. We explored the utility of routine histopathology and compared immunohistochemistry (IHC) and in situ hybridization (ISH) in pneumonic bovine lung tissue samples for the detection of M. bovis infection. Samples were analyzed for M. bovis using mycoplasma bacterial culture (screening test), H&E staining, IHC, and ISH. We found that “compatible histologic lesions” are not entirely predictive of the presence of M. bovis on culture, IHC, or ISH, and also that there was no statistical difference between IHC and ISH for detecting M. bovis. We conclude that IHC and ISH can be used interchangeably to detect M. bovis infections in pneumonic bovine lung.

Keywords

bronchopneumonia bovine respiratory disease complex pneumonia

The bovine respiratory disease complex (BRDC) is a disease of great economic importance that involves environmental and nutritional factors, a weakened host immune system, and the participation of several viral and bacterial pathogens.⁷ The bacterial pathogens most commonly associated with BRDC are Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis (Mycoplasma) bovis.^5,7,12 Common viruses involved in BRDC include bovine alphaherpesvirus 1, bovine respiratory syncytial virus, and bovine parainfluenza-3 virus. While the role of M. bovis in BRDC remains poorly understood, this bacterium is frequently detected in cases of pneumonia in juvenile cattle in southwest Virginia, prompting the need for a more thorough investigation of its involvement in this complex.³

M. bovis and other mycoplasmas typically inhabit mucosal surfaces, such as those of the respiratory, urogenital, and gastrointestinal tracts, as well as the conjunctiva and mammary gland.^6,13,14 M. bovis colonizes the upper respiratory tract of calves early in life and is typically a secondary opportunistic pathogen, often contributing to polymicrobial infections in the lung.^4,10,12,18 M. bovis is often found in large numbers in the lungs of cattle with BRDC, most likely due to impairment of the mucociliary apparatus.²² Gross lesions in the lung caused by M. bovis are those of a caseonecrotic bronchopneumonia. Histologically, the affected lung has conspicuous round-to-irregular areas of caseous necrosis within airways surrounded by well-delineated areas of granulation tissue.^11,12

M. bovis can be detected with several techniques. Microbial culture can be used to grow Mycoplasma spp. with their characteristic fried-egg appearance, but speciation within the genus requires additional testing.^8,17 PCR can be used to detect the pathogen through amplifying bacterial DNA from samples such as lung, nasal swabs, and bronchoalveolar lavage.¹⁶ Histopathology, immunohistochemistry (IHC), and in situ hybridization (ISH) can also be used to detect M. bovis.^1,9,16 Histopathology detects the lesions produced by M. bovis, providing a presumptive diagnosis but does not confirm the infection. IHC detects and localizes M. bovis proteins within tissue; ISH detects and localizes M. bovis DNA in tissue.^1,9 We explored the utility of histopathology for the detection of lesions compatible with M. bovis infection and compared IHC and ISH for detection of M. bovis in lung tissue samples collected from bovine bronchopneumonia cases.

Materials and methods

Case selection

In our retrospective study, we used tissue samples collected from animals submitted to the Wytheville Regional Animal Health Laboratory, Virginia Department of Agriculture and Consumer Services (Wytheville, VA, USA) in 2020. Fifteen cases were selected from 1–12-mo-old animals with bronchopneumonia from a previous pilot study. By a Mycoplasma spp. culture (screening test), 10 of 15 cases were positive and 5 were negative. Briefly, fresh lung specimens collected from the right cranial lobe were used for routine aerobic culture (Table 1) and also inoculated onto mycoplasma agar with cefoperazone (Hardy) and incubated in a humidity chamber at 37°C in a 5% CO₂ incubator, for up to 7 d. Mycoplasma spp. were identified by colony morphology and Diene stain. In addition, sections from the right cranial lung lobe were collected in formalin and processed routinely for histopathology. One lung block per animal was analyzed. Deep recuts of each lung block were obtained and evaluated side-by-side with the 3 detection methods (routine H&E, IHC, and ISH).

Table 1.

Aerobic bacteria isolated from the lung of each case of bronchopneumonia included in our study.

Case	Isolate 1	Isolate 2
1	Mannheimia haemolytica	Trueperella pyogenes
2	Mannheimia haemolytica
3	Pasteurella multocida
4	Pasteurella multocida
5	Pasteurella multocida
6	Pasteurella multocida	Trueperella pyogenes
7	Trueperella pyogenes
8	No growth
9	No growth
10	Pasteurella multocida
11	Pasteurella multocida	Trueperella pyogenes
12	Mannheimia haemolytica
13	Moraxella osloensis
14	No growth
15	Pasteurella multocida

Histopathology

We evaluated the diagnostic value of histopathology using H&E-stained slides by carefully examining each slide for the presence or absence of lesions compatible with M. bovis infections (M. Brown, Z. Laney, L. Tabatabai, F. Carvallo). We defined “compatible lesions” as lung sections with one or more rounded areas of caseous necrosis within the airways, surrounded by a mixed inflammatory infiltrate and fibrosis (Fig. 1A). A slide was categorized as positive when one or more compatible lesions were present, or as negative, when no compatible lesions were found.

Figure 1.

“Compatible lesions” of Mycoplasmopsis (Mycoplasma) bovis infection in calves with pneumonia. A. A round structure (presumptive bronchiole) with abundant eosinophilic necrotic cellular debris in the center, surrounded by fibrous connective tissue containing neutrophils and macrophages. H&E. B. Deeper section of the same bronchiole, with immunopositivity along the margins of the lesion, between the necrotic cellular debris and the cellular component. M. bovis immunohistochemistry, diaminobenzidine chromogen, hematoxylin counterstain. C. A strong signal along the margins of the necrotic cellular debris and the cellular component. M. bovis in situ hybridization, mRNA RED probe amplification kit, hematoxylin counterstain. Bars = 100 µm.

Immunohistochemistry

Following automated deparaffinization of formalin-fixed, paraffin-embedded (FFPE) tissue sections (EZ Prep 1X; Roche), antigen retrieval was accomplished with an application of Cell Conditioning 1 (CC1; Roche) solution for 16 min at 100°C. Endogenous peroxidases were blocked with an application of Inhibitor CM (Roche) for 8 min. The M. bovis primary antibody (1:800, pool MYB57, MYB87, MYB163; obtained from Dr. R. Rosenbusch, Iowa State Diagnostic Laboratory, 2005)¹ was then applied for 28 min at 37°C and detected with a conjugated multimer (OmniMap anti-rabbit HRP multimer, Roche; 16 min). A standard application of each of Discovery ChromoMap DAB (Roche), hematoxylin (8 min), and bluing reagent (4 min) completed the staining process. Slides were removed from the automated IHC stainer (Discovery Ultra; Roche) and automatically dehydrated and coverslipped (Tissue-Tek Prisma Plus stainer and film coverslipper; Sakura). A positive IHC result was identified as brown granular staining within the tissue sections (Fig. 1B). Positive controls were obtained from the California Animal Health and Food Safety Laboratory (San Bernardino, CA, USA), from a calf with M. bovis infection confirmed by positive PCR and culture.

In situ hybridization

For cellular localization of M. bovis nucleic acid in FFPE tissue sections, a validated ISH method (RNAscope; Advanced Cell Diagnostics) was performed using a standard protocol and an automated ISH research platform (Discovery Ultra; Roche). After deparaffinization and target retrieval (cat. 760-248, mRNA sample prep kit; Roche), 4-μm tissue sections mounted on charged slides were hybridized with 200 μL of a 14ZZ ISH probe targeting a 1,660-nucleotide region of the M. bovis UVRC gene encoding a deoxyribodipyrimidine photolyase (GenBank AF003959.1; 508289, B-M. bovis-UVRC, Advanced Cell Diagnostics). This was followed by signal amplification (cat. 760-236, mRNA RED probe amplification kit; Roche), detection (cat. 760-234, mRNA RED detection kit; Roche), and counterstaining with hematoxylin. Positive signal was identified as red staining within infected cells (Fig. 1C).

Grading of intensity of IHC and ISH staining

We evaluated the intensity of IHC and ISH staining using nonparametric and subjective scales of 0–3 (Fig. 2; M. Brown, Z. Laney, L. Tabatabai, F. Carvallo): 0 = negative staining, no staining could be found; 1 = mild intensity, staining was present but only faintly visible or in clusters using 10× magnification; 2 = moderate intensity, staining could be seen using 4× magnification, but the intensity was variable, and staining identification required more extensive searching across the slide; 3 = strong intensity, the staining was intense across most of the lesions, and the stain could easily be visualized using 4× magnification (Fig. 2). Intensity values were also separated between 2 histologic locations in the lung: distal airways (bronchioles) and alveoli.

Figure 2.

Grading of immunohistochemistry for Mycoplasmopsis (Mycoplasma) bovis. A. Grade 0 = no staining could be found. B. Mild intensity or grade 1 = staining, but in clusters and faintly visible using 10× magnification. C. Moderate intensity or grade 2 = staining could be seen using 4× magnification, but the intensity was variable, and identification of staining required more extensive searching across the slide. D. Strong intensity or grade 3 = intensity was strong across most of the visible lesions on the slide, and the stain could very easily be visualized using 4× magnification. Hematoxylin counterstain in all slides. Bars = 100 µm.

Statistical analysis

A weighted kappa statistical method was used to test for agreement between the 2 grading scales used to evaluate the intensity of IHC and ISH staining. A value of 0 = agreement equivalent to chance, <0.2 = slight agreement, 0.21–0.40 = fair agreement, 0.41–0.60 = moderate agreement, 0.61–0.80 = good agreement, 0.81–1.00 = very good agreement, and 1 = perfect agreement between the 2 scales.²⁰

Results

Of the 5 of 15 animals that were negative on mycoplasma culture, 4 did not have H&E-compatible lesions and 1 animal did have H&E-compatible lesions (Table 2). Of the 5 samples with no mycoplasma-positive culture, M. bovis was not detected through IHC or ISH. In case 11, abundant acid hematin was present in IHC and ISH samples. Of the 10 samples with positive mycoplasma culture, 7 had M. bovis–compatible lesions with H&E, and 3 did not have compatible lesions. In all 10 samples with positive mycoplasma culture, M. bovis was detected with IHC and ISH in either airway and/or alveolar areas of the lung (Fig. 3). Histologic findings in alveolar areas included neutrophils and macrophages, necrotic cellular debris, and small amounts of fibrin. The alveolar interstitium was variably expanded with similar inflammatory infiltrates and occasional thrombi. M. bovis was detected in 7 samples in both the airway and alveolar areas of the lung, in 2 samples only in the airways, and in 1 sample only in alveoli. In total, using IHC and ISH, M. bovis was detected in airways in 9 samples and in alveoli in 8 samples.

Table 2.

Scoring of immunohistochemistry (IHC) and in situ hybridization (ISH) results for Mycoplasmopsis (Mycoplasma) bovis bovine bronchopneumonia cases positive by mycoplasma culture (screening test).

Case	Mycoplasma spp. cultured	H&E compatible lesions	IHC grade		ISH grade
Case	Mycoplasma spp. cultured	H&E compatible lesions	Airways	Alveoli	Airways	Alveoli
1	Yes	Yes	3	3	1	3
2	Yes	Yes	3	3	3	3
3	Yes	Yes	2	3	3	3
4	Yes	Yes	2	3	2	3
5	Yes	Yes	3	3	3	3
6	Yes	Yes	1	0	1	0
7	Yes	Yes	2	0	1	0
8	Yes	No	2	3	3	3
9	Yes	No	2	1	3	2
10	Yes	No	0	3	0	3
11	No	Yes	0	0	0	0
12	No	No	0	0	0	0
13	No	No	0	0	0	0
14	No	No	0	0	0	0
15	No	No	0	0	0	0

M. bovis detection intensity grading system for both IHC and ISH: 0 = absent; 1 = mild; 2 = moderate; 3 = strong.

Figure 3.

Alveolar detection of Mycoplasmopsis (Mycoplasma) bovis. A. In this area, a dense inflammatory infiltrate in alveoli is composed of neutrophils and macrophages, together with necrotic cellular debris. H&E. B. A deeper section of the same topographic area, with the presence of M. bovis immunopositivity in alveolar inflammatory cells and free in the alveolar lumens. M. bovis immunohistochemistry, diaminobenzidine chromogen, hematoxylin counterstain. C. Deeper section of the same topographic area, with a strong signal in inflammatory cells and free in the alveolar lumens. M. bovis in situ hybridization, mRNA RED probe amplification kit, hematoxylin counterstain. Bars = 50 µm.

The weighted kappa for agreement of IHC and ISH in airways is 0.71 (95% CI: 0.49–0.93%; Table 3), which indicates good agreement. M. bovis was not detected by ISH in any of the 6 cases with no immunoreactivity for M. bovis on IHC. Among subjects with immunoreactivity for M. bovis by IHC, there was variable and partial agreement in grading of M. bovis by ISH.

Table 3.

Comparison of immunohistochemistry (IHC) grade to in situ hybridization (ISH) grade for Mycoplasmopsis (Mycoplasma) bovis in the airways of lungs from calves with bronchopneumonia.

IHC	ISH
IHC	Not present	Mild	Moderate	Strong
Not present	6	0	0	0
Mild	0	1	0	0
Moderate	0	1	1	3
Strong	0	1	0	2

The weighted kappa for the agreement of IHC and ISH alveoli is 0.95 (95% CI: 0.87–1.00%; Table 4), which indicates very good agreement. All 7 patients with strong immunoreactivity for M. bovis on IHC also had strong detection of M. bovis by ISH. M. bovis was not detected by ISH in any of the 7 cases with no immunoreactivity for M. bovis on IHC.

Table 4.

Comparison of immunohistochemistry (IHC) grade to in situ hybridization (ISH) grade for Mycoplasmopsis (Mycoplasma) bovis in the alveoli of lungs from calves with bronchopneumonia.

IHC	ISH
IHC	Not present	Mild	Moderate	Strong
Not present	7	0	0	0
Mild	0	0	1	0
Moderate	0	0	0	0
Strong	0	0	0	7

Discussion

We found suboptimal sensitivity in identifying calves with Mycoplasma spp. in the lung when relying on H&E staining alone; we detected lesions in only 7 of 10 culture-positive cases. Studies focusing on histologic lesions associated with M. bovis pneumonia have found variable reliability associated with compatible lesion presentation. In one study, 18 of 18 naturally infected cattle had compatible histologic lesions¹¹; an earlier study only demonstrated such lesions in 12 of 25 cases confirmed positive for M. bovis via culture.² Therefore, it is not a guarantee that H&E-compatible lesions are present in all cases from which Mycoplasma spp. are cultured. On the other hand, we found compatible lesions in one case with negative Mycoplasma spp. culture, which indicates either that culture is a less sensitive method or that compatible lesions could also be identified in cases caused by agents other than M. bovis, such as M. haemolytica and H. somni.¹²

The use of IHC for the detection of M. bovis in FFPE bovine lung tissue samples has been reported in several studies,^2,11,21 which demonstrated that IHC had value in identifying these infections. On the other hand, the use of ISH for the detection of M. bovis has only been reported once in an aborted bovine fetus and a neonatal calf, where it was shown to be an effective detection technique.⁹

We found good agreement between IHC and ISH for the detection of M. bovis in airways and very good agreement for detection in alveoli. Therefore, IHC and ISH could be used interchangeably for reliable detection of M. bovis in bovine lung tissue. However, we only had a few cases in the mild-to-moderate categories; IHC and ISH may be equivalent at detecting strongly positive or negative cases but may not perform as well in detecting less distinct lesions or lower bacterial loads. Larger validation studies are needed to confirm these findings and rule out these concerns.

We detected M. bovis in the airways by IHC and ISH in 9 of 10 cases in which Mycoplasma spp. were isolated. M. bovis antigen and DNA were particularly abundant within the bronchioles. This finding agrees with the expected localization of M. bovis within the lungs, as the earliest lesions are believed to begin in small airways, especially bronchioles.⁵ Compatible lesions identified on H&E staining were detected by IHC and ISH as well. Only case 10 had a positive mycoplasma culture but absence of any lesions within the airways, as well as absence of any compatible lesions detected on H&E staining. We hypothesize that the lesion was missed in the analyzed section, and that this may be a case of more acute M. bovis infection or of coinfection with another bacterium.

Regarding the detection of M. bovis in the rest of the pulmonary parenchyma, 8 of 10 samples in which Mycoplasma spp. were cultured had IHC and ISH positivity within alveolar spaces. Case 10 had M. bovis detected within alveoli only. In cases 2, 8, and 10, IHC and ISH positivity was within foci of coagulative necrosis, and alveolar architecture was still discernable within the lesion. Previous studies have demonstrated M. bovis both on the surface of alveolar epithelium and within alveolar macrophages and neutrophils.^8,21 It has been postulated that areas with coagulative necrosis can also be found with M. bovis infection, but it is not clear if this lesion is a transitional form to caseonecrotic “compatible” lesions or if coagulative necrosis and caseonecrotic foci are 2 separate lesions.^2,11 It has also been theorized that the lesions are distinct and may be due to different strains of M. bovis or due to coinfection with other pathogens.^11,21 We corroborate that there are 2 sets of lesions caused by M. bovis infection: 1) the compatible lesion, which is likely due to bronchiolar necrosis, chronic inflammation, and granulation tissue, and 2) irregular areas of acute necrosis, which first manifest as small foci of inflammation in alveoli that may progress into tortuous areas of coagulative necrosis in the pulmonary parenchyma. Areas of coagulative necrosis do resemble lesions caused by other pathogens associated with BRDC, thus the interpretation of coagulative necrosis should be made with caution.¹² We also conclude that routine H&E stain may serve as an initial screening for the detection of M. bovis, but the final detection must be performed with an additional confirmatory test, such as culture, PCR, IHC, or ISH. Also, the absence of compatible lesions is not absolute evidence of negative M. bovis status.

We found some challenges and limitations during our study. The interpretation of the IHC sample from case 11 was complicated by the presence of acid hematin. Acid hematin is a brown-to-black amorphous pigment that is produced by an acid acting on hemoglobin in the sample when the pH of the formalin that is used to fix the sample is low.¹⁹ The color and shape of this pigmented material closely resemble positive signaling of M. bovis in IHC revealed with diaminobenzidine, making interpretation of the samples more difficult and increasing chances for an incorrect clinical diagnosis. In addition, thrombi within blood vessels were present in a few samples (cases 1, 3, and 11). Thrombosis may resemble the rounded areas of caseous necrosis caused by M. bovis, which can be confused as a compatible lesion by an inexperienced eye. Another limitation is the fact that our mycoplasma cultures were not followed up by speciation within the genus, so it is impossible to know if all of the animals with positive mycoplasma cultures were in fact M. bovis. There are 13 other Mycoplasma spp., such as M. bovirhinis, that could also be detected with Mycoplasma culture.¹⁵ However, our IHC and ISH procedures were specific for M. bovis. It is also possible that areas of coagulative necrosis due to early infection with M. bovis were missed in some sections that included only compatible or more chronic lesions. We suggest that the analysis of M. bovis–associated lesions should always use a confirmatory test, and we recognize that acute lesions can be confused with other bacterial infections. Another limitation of our study is the unknown status of viral infectious causes of pneumonia in the analyzed specimens.

Footnotes

Acknowledgements

We thank Jinhua Zhang and Daren Lewis for their assistance in standardizing our ISH methods, and Rachel Derscheid from Iowa State University for her technical support with IHC testing.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This project was funded by the Office of Research and Graduate studies RGS130532, Virginia Maryland College of Veterinary Medicine and the USDA National Institute of Food and Agriculture, Animal Health and Disease project 7004199.

ORCID iDs

Francisco A. Uzal

Francisco R. Carvallo

References

Adegboye

, et al Monoclonal antibody-based immunohistochemical technique for the detection of Mycoplasma bovis in formalin-fixed, paraffin-embedded calf lung tissues. J Vet Diagn Invest 1995;7:261–265.

Adegboye

, et al Immunohistochemical and pathological study of Mycoplasma bovis-associated lung abscesses in calves. J Vet Diagn Invest 1995;7:333–337.

Carvallo , et al Real time PCR for the detection of Mycoplasma bovis and viral pathogens of calf pneumonia in southwest Virginia based on formalin-fixed paraffin embedded specimens. In: Proc ACVP/ASVCP Annual Meeting; Chicago, IL; Oct 2023.

Caswell

, et al Mycoplasma bovis in respiratory disease of feedlot cattle. Vet Clin North Am Food Anim Pract 2010;26:365–379.

Caswell

Williams

. Respiratory system. In: Maxie

, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. 6th ed. Vol. 2. Elsevier, 2016:465–591.

Griffin

, et al Bacterial pathogens of the bovine respiratory disease complex. Vet Clin North Am Food Anim Pract 2010;26:381–394.

Grissett

, et al Structured literature review of responses of cattle to viral and bacterial pathogens causing bovine respiratory disease complex. J Vet Intern Med 2015;29:770–780.

Hermeyer

, et al Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: characterization of lung pathology, persistence of variable surface protein antigens and local immune response. Acta Vet Scand 2012;54:9.

Hermeyer

, et al Demonstration of Mycoplasma bovis by immunohistochemistry and in situ hybridization in an aborted bovine fetus and neonatal calf. J Vet Diagn Invest 2012;24:364–369.

10.

Holman

, et al The nasopharyngeal microbiota of feedlot cattle. Sci Rep 2015;5:15557.

11.

Khodakaram-Tafti

López

Immunohistopathological findings in the lungs of calves naturally infected with Mycoplasma bovis. J Vet Med A Physiol Pathol Clin Med 2004;51:10–14.

12.

López

Martinson

. Respiratory system, thoracic cavities, mediastinum and pleurae. In: Zachary

, ed. Pathologic Basis of Veterinary Disease. 7th ed. Elsevier, 2022:547–642.

13.

Maunsell

Donovan

GA.

Mycoplasma bovis infections in young calves. Vet Clin North Am Food Anim Pract 2009;25:139–177.

14.

Maunsell

, et al Mycoplasma bovis infections in cattle. J Vet Intern Med 2011;25:772–783.

15.

McAuliffe

, et al 16S rDNA PCR and denaturing gradient gel electrophoresis; a single generic test for detecting and differentiating Mycoplasma species. J Med Microbiol 2005;54:731–739.

16.

Nicholas

Ayling

RD.

Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 2003;74:105–112.

17.

Parker

, et al A review of mycoplasma diagnostics in cattle. J Vet Intern Med 2018;32:1241–1252.

18.

Perez-Casal

Pathogenesis and virulence of Mycoplasma bovis. Vet Clin North Am Food Anim Pract 2020;36:269–278.

19.

Perino

, et al Diagnostic guidelines for the histological particle algorithm in the periprosthetic neo-synovial tissue. BMC Clin Pathol 2018;18:7.

20.

Ranganathan

, et al Common pitfalls in statistical analysis: measures of agreement. Perspect Clin Res 2017;8:187–191.

21.

Rodríguez

, et al Pathological and immunohistochemical studies of natural and experimental Mycoplasma bovis pneumonia in calves. J Comp Pathol 1996;115:151–162.

22.

Timsit

, et al Distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia. Vet Microbiol 2018;221:105–113.