Bronchopneumonia with interstitial pneumonia

in beef feedlot cattle

Luke Haydock1, Kent Fenton2, Ruud Veldhuizen3, Dani Smerek2, Lauren Sergejewich1, David Renaud1, Davor Ojkic1, Jeff Caswell1

1 University of Guelph

2 Feedlot Health Management Services

3 University of Western Ontario

1

https://hdl.handle.net/10214/27003

BSVP, 10 Jun 2022

Interstitial lung disease: overview

• Disease targeting lung interstitium, typically alveolar septa

• Diffuse alveolar damage:

• Pathophysiology: effects on gas exchange, stiffness / compliance, permeability of air-blood barrier, dryness of alveoli, surfactant function

• Morphology: edema, hyaline membranes, type II pneumocyte proliferation, fibrosis

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Causes of interstitial (or bronchointerstitial) lung disease

• Ingested toxicants: 3-methylindole (tryptophan, lush pasture), mouldy sweet potatoes (ipomeanol), perilla ketones (purple mint)

• Inhaled gases: nitrogen dioxide / silo gas, chlorine gas

• Viruses

• Larval migration: Dictyocaulus, Ascaris suum

• Sepsis & SIRS

• Hypersensitivity: milk allergy, hypersensitivity to lungworms?

• Surfactant dysfunction (prematurity, genetic)

• Heart failure, acid aspiration, smoke inhalation, ventilator-induced, adverse drug reaction, lobe torsion, reperfusion injury, pancreatitis, uremia, strangulation, near-drowning, contusions

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Acute interstitial pneumonia (AIP, fog fever) in pastured cattle

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Ingestion of L-tryptophan

Deamination to indoleacetic acid (IAA) by ruminal

microflora

Decarboxylation of IAA to 3-MI by

Lactobacillus spp.

Absorption of 3-MI into

bloodstream

Dehydrogenation of 3-MI to 3-Methyleneindolenine

(3-MEIN) by pulmonary cytochrome P450s

3-MEIN DNA adducts in type I

and II pneumocytes

Cell death and diffuse alveolar

damage

Dickinson, 1967

Yokoyama et al., 1977; Carlson and Breeze, 1984

Gram, 1997

Ruangyuttikarn et al., 1992

See Haydock et al, Animal Health Research Reviews, in press

“Acute/atypical interstitial pneumonia” of feedlot cattle • Acute, sporadic • Larger feedlots • Spring & summer; hot & dusty • Deaths late in the feeding period • Well-conditioned yearlings • ∼3x heifer predominance

• 2.8% incidence (NAHMS, USDA)

• ↑ plasma 3-methylindole concentration in plasma, ↓ urine 3-MI metabolite; no difference in 3-methyleneindolenine in lung tissue (Ayroud, 2000)

• ↑ 3-methyleneindolenine in lung and blood (Loneragan, 2001)

• Possible role of subclinical rumen acidosis … Lactobacillus … 3-MI metabolism

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Credit: Dr. Kent Fenton + Dani Smerek. Feedlot Health Management Services, Okotoks, AB

“BIP” Bronchopneumonia with interstitial pneumonia

aka bronchointerstitial pneumonia

• Gross pathological diagnosis based on postmortem findings of:

• Lesions of bronchopneumonia (BP) in cranioventral lung

• Lesions of interstitial pneumonia (IP) in caudodorsal lung

• Concurrent bronchopneumonia (i.e. BIP) reported in 32-97% of feedlot AIP cases (Curtis et al, 1979; Hjerpe, 1983; Sorden et al., 2000; Woolums et al., 2004; Panciera and Confer, 2010; Valles et al., 2015).

• Chance concurrence, or a distinct disease?

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Hypothesis

• Bronchopneumonia with interstitial pneumonia is a distinct disease entity with a unique pathogenesis.

Objective

• 1. Characterize the pathological, microbiological and epidemiological features of BIP to distinguish it as a distinct disease entity.

• 2. Provide insights into the features of BIP pathogenesis that differentiate it from similar diseases (BP, AIP).

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Overview

• BIP characterization

• Accuracy of gross diagnosis

• Histopathology

• Microbiology

• Epidemiology study

• Investigation of BIP pathogenesis

• Cytokine levels in serum (IL-1β, IL-6, IL-8) Role of hyperinflammation / “cytokine storm” ?

• Surfactant analysis Role of altered surfactant composition or function?

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Methods

• Case selected: • Mortalities on commercial Western Canadian beef feedlots.

• Convenience samples, January 2018 to January 2022

• BIP (n = 18), BP (n = 24) and AIP (n = 13) mortalities

• Gross Diagnosis: • Postmortems by feedlot vet or trained technician

• Standardised photographs including right lung in situ, cut surface of cranioventral and caudodorsal lung

• Diagnosis by a single experienced feedlot veterinarian based on PM photos

• Final diagnoses by histopathology (“gold standard”)

• Sample collection in each case: • Histopathology: cranioventral & caudodorsal lung, 3 each

• Bacterial and mycoplasma culture: cranioventral & caudodorsal lung, 3 each

• Virology ([RT-]qPCR: caudodorsal lung, 3 each

• Heart blood serum

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BIP: bronchopneumonia with interstitial pneumonia

BP: bronchopneumonia

AIP: acute/atypical interstitial pneumonia

Results: accuracy of gross diagnosis

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6 BP 4 AIP

2 BP 1 AIP

Results: quantification of bronchopneumonia

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65.7 %

Proportion of Lung Grossly Affected by Bronchopneumonia

% 95 % Confidence Interval (%)

BIP 53 45 – 61

BP 45 26 – 53 P = 0.21

Mean DOF

Mean BW at death (kg)

P value

BIP 127 349 0.021

BP 89 287 0.023

Methods: characterisation of lesions

• Blinded to gross diagnosis; review and scoring of histologic lesions

• Aging of lesions:

• Acute: no fibrosis

• Subacute: immature granulation tissue

• Chronic: mature fibrosis

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15

16

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Normal

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19

20

21

CK

Mac387

HE

Iba1

22

CK

Mac387

HE

Iba1

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Masson

Mac387

HE

CK

Results Histological

Characterization - Lesion Prevalence

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Hyaline Membranes

Results

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Histological Characterisation – Chronicity of BIP

P < 0.001

Methods

• Bacterial culture: cranioventral and caudodorsal lung, liver and

kidney

• Mycoplasma culture: cranioventral and caudodorsal lung

• Virology PCR: BHV-1, BPIV3, BRSV, BCV, bovine adenovirus 1-8;

caudodorsal lung

• High-throughput sequencing on caudodorsal lung of 3 BIP and 3

BP mortalities (Université de Montréal)

• Bovine hokovirus PCR designed in-house following detection of

bovine hokovirus genome sequences by high-throughput

sequencing 26

Microbiological Characterisation (Animal Health Laboratory)

Results Microbiological Characterisation

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* P < 0.05 * P < 0.05

a BIP (n = 12), BP (n = 20), AIP (n = 10)

Methods Epidemiological Characterisation

• Study design: • Cross-sectional study of all mortalities occurring on 4 Canadian

commercial beef feedlots from Jan 2016 – Jan 2020.

• Feedlots included: high prevalence of BIP; standardized post-mortem protocol with photos taken and reviewed by a feedlot vet.

• Database: • Animal health and management data: 9909 mortalities (1105 BIP).

• Data included: sex, age class, days on feed, arrival weight, death weight, vaccination status, treatments, date of arrival, date of death and mortality diagnosis

• Data analysis: • BIP vs AIP, BIP vs BP, BIP vs Other BRD, BIP vs Non-BRD.

• Chi-square (X2) test for categorical data.

• Independent sample T-test for numerical data.

• Multivariate logistic regression to determine most significant predictors of BIP mortality.

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Results Epidemiological Characterisation – Prevalence of Mortality

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Post-Mortem

Diagnosis

Percentage of Study

Population (%)

BIP 11.2

BP 17.4

AIP 8.9

Other BRD 1.3

Non-BRD 61

Expected prevalence of concurrent BP and AIP (i.e.

incidental BIP) = 5.7 %

Actual prevalence of BIP = 11.2 %

BIP occurs at a rate 1.96-fold higher than the expected

rate of incidental BP and AIP co-occurrence

Results Epidemiological Characterisation – Average BIP Mortality:

• Is a steer

• Odds Ratio (OR) = 1.23

• Spends 3 – 4 months on the feedlot before dying.

• Days on feed = 115 days

• Is poorly thriving

• Daily weight gain = 0.46 kg / day

• Postmortem weight = 334 kg

• Has a history of chronic / recurrent illness.

• Time to death from first illness = 45 days

• 2 illnesses (OR = 1.99); 3 illnesses (OR = 3.17); 4+ illnesses (OR = 3.35)

• Dies in winter (OR = 1.69).

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Results

Epidemiological Characterisation – Sex Distribution

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BIP mortalities had a distinct sex distribution compared to BP and AIP (P < 0.001)

Results Epidemiological Characterisation –

Number of Treatments Prior to Death

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BIP mortalities experienced significantly (P < 0.004) more episodes of illness prior to death compared to BP and AIP.

Results Epidemiological Characterisation – Seasonality

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BIP BP AIP

Fall 14 17 13

Winter 48 54 24

Spring 26 21 28

Summer 12 7.9 35

TOTAL 100 100 100

P-value < 0.001 < 0.001

Percentage of Mortalities by Season (%)

BIP mortalities have a mid-winter to early-spring seasonal distribution that is distinct from that of AIP mortalities (P < 0.001)

Results Epidemiological Characterisation – Comparison to BP and AIP

• Compared to BP, cattle dying of BIP have significantly (P < 0.001): • HIGHER weights at time of death – Avg. 334 kg vs 295 kg.

• MORE days on feed at time of death – Avg. 115 days vs 81 days.

• HIGHER average daily weight gain prior to death – Avg. 0.46 kg/day vs 0.09 kg/day.

• LONGER time to death from the first signs of clinical illness – Avg. 45 days vs 33 days.

• Compared to AIP, cattle dying of BIP have significantly (P < 0.001): • LOWER weights at time of death – Avg. 334 kg vs 469 kg.

• FEWER days on feed at time of death – Avg. 115 days vs 185 days.

• LOWER average daily weight gain prior to death – Avg. 0.46 kg/day vs 1.12 kg/day.

• LONGER time to death from the first signs of clinical illness – Avg. 45 days vs 26 days. 34

Results Epidemiological Characterisation – Logistic Regression

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Methods BIP Pathogenesis – Cytokine Analysis

Does hyperinflammation / “cytokine storm” contribute to BIP?

• Cytokine ELISA

• Concentration of IL-1β, IL-6, and IL-8 quantitatively measured by commercially available bovine assays (Invitrogen, Thermo Fisher Scientific; Mabtech).

• Assays performed on heart blood serum from BIP (n = 5), BP (n = 5), and AIP (n = 5) mortalities.

• Mortalities were approximately matched for days on feed.

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BIP Pathogenesis – Cytokine Analysis in Serum

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There was no significant (P > 0.05) difference in heart blood serum concentrations of IL-1β, IL-6, or IL-8 between BIP, BP, and AIP mortalities.

Analysis of postmortem heart blood serum: BIP (n = 5), BP (n = 5), and AIP (n = 5) Mortalities were roughly matched (where possible) for days on feed.

41 Heifer, died after intramuscular LPS injection

Surfactant Overview

• Complex mixture of phospholipids and surfactant proteins,

synthesized and secreted by type II pneumocytes • Lowers alveolar surface tension: alveolar stability during ventilation

cycles • Altered surfactant function in diffuse alveolar damage (eg NRDS,

ARDS) • Two main extracellular components:

• Large aggregates – functional, surface-active fraction • Small aggregates

• Lung injury / inflammation modifies surfactant function • Leakage of plasma proteins into alveoli • Incorporation of surfactant components into polymerizing fibrin • Inflammation: degradation and inhibition of surfactant

components 42

Methods BIP Pathogenesis –

Altered surfactant composition / function in BIP?

• Samples

• Bronchoalveolar lavage (BAL) fluid collected at postmortem from BIP (n = 4), BP (n = 8), and AIP (n = 1) mortalities

• Control BAL from 6 normal yearling cattle:

• immediately after slaughter (Nfresh).

• after 24 hours of refrigeration (N24 hours)

• Analysis in Veldhuizen lab, University of Western Ontario

• Surfactant isolated from BAL fluid; composition & function tested

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Methods BIP Pathogenesis – Surfactant Analysis

Does altered surfactant composition / function contribute to BIP?

• Surfactant function

• Surface tension-reducing capabilities of surfactant assessed with a constrained sessile drop surfactometer.

• A surfactant drop is applied to pedestal with a 1 mm pinhole.

• The drop is then cycled through periods of compression and expansion.

• Images of the surfactant drop are taken during compression-expansion cycling.

• The minimum surface tension during each cycle is then determined by means of image analysis with axisymmetric drop shape analysis software.

• Similar analysis also performed on “extracted” surfactant from BIP (n = 4), BP (n = 4), and Normal (n = 4) cattle.

• Surfactant extraction involves separation of surfactant lipids and surfactant proteins-B and -C from the original surfactant fraction.

• This removes the inhibitory effects of plasma / inflammatory proteins on surfactant function.

• But also, the beneficial effects of surfactant protein-A are lost.

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Results BIP Pathogenesis – Surfactant Composition

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In BIP surfactant vs control, increased total protein concentration and possibly reduced proportion of large aggregates (P < 0.05)

Results

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AIP (n=1)

BP (n=8) BIP (n=4)

Nfresh (n=6) N24h (n=5)

1. BIP: Reduced surfactant function (increased minimum surface tensions during compression/ expansion cycles)

BIP Pathogenesis – Surfactant Function

Results BIP Pathogenesis – Surfactant Function

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2. BIP: proteins in surfactant do not significantly contribute to reduced surfactant function during compression / expansion cycles (X = 1 – 20)

Results BIP Pathogenesis – Surfactant Function

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3. Adsorption, formation, and/or stabilization of the initial surfactant film (X = 0) is negatively affected by proteins in surfactant

Limitations

• Epidemiology

• Group classification (i.e. BIP, BP, AIP) not validated by histopathology (but, similar epidemiology findings in the 55 histologically confirmed diagnoses)

• Surfactant analysis

• Low numbers. N=1 for AIP.

• Extracted surfactant: also removes surfactant protein-A

• Results do not differentiate cause and effect

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Conclusions – Pathology and Microbiology

1. BIP is a unique disease entity that occurs in beef feedlot cattle (and dairy-beef)

2. Despite a distinctive gross appearance, accurate diagnosis of BIP requires histological evaluation of both cranioventral and caudodorsal lung

3. In BIP, bronchopneumonia in the cranioventral lung is typically chronic, and interstitial lung injury in the caudodorsal lung is typically acute

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Conclusions – Epidemiology

1. BIP prevalence is ∼2-fold higher than the prevalence expected from chance concurrence of BP and AIP

2. Typical BIP cases occur in winter and affect steers, poorly thriving, history of chronic/recurrent illness, and die 3 – 4 months after arriving on the feedlot

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Conclusions – BIP Pathogenesis

1. No differences in serum IL-1β, IL-6, IL-8, suggesting that hyperinflammation / “cytokine storm” does not contribute to pathogenesis

2. In BIP, lung surfactant has a reduced function for lowering surface tension, increased proteins in surfactant impede formation of the initial surfactant film in an in vitro setting, but reduced surfactant function during dynamic compression/expansion cycles involves a mechanism other than inhibition by proteins

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Acknowledgments

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University of Guelph Luke Haydock Lauren Sergejewich Dave Renaud Davor Ojkic Feedlot Health Management Services Kent Fenton Dani Smerek Univ. of Western Ontario Ruud Veldhuizen Linda McCaig