Veterinary Microbiology 264 (2022) 109271

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Replication of Streptococcus equi subspecies zooepidemicus infection in swine

Samantha J. Hau a, Kristina Lantz b, Keira L. Stuart b, Panchan Sitthicharoenchai c,
Nubia Macedo c, Rachel J. Derscheid c, Eric R. Burrough c, Suelee Robbe-Austerman b,
Susan L. Brockmeier a, *
a
Virus and Prion Research Unit, National Animal Disease Center, ARS, USDA, Ames, IA, United States
b
National Veterinary Services Laboratories, APHIS, USDA, Ames, IA, United States
c
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Streptococcus equi subspecies zooepidemicus (SEZ) is a commensal bacterium of horses and causes infections in
Streptococcus equi ssp zooepidemicus mammalian species, including humans. Historically, virulent strains of SEZ caused high mortality in pigs in China
Swine and Indonesia, while disease in the U.S. was infrequent. More recently, high mortality events in sows were
Septicemia
attributed to SEZ in North America. The SEZ isolates from these mortality events have high genetic similarity to
High mortality
an isolate from an outbreak in China. Taken together, this may indicate SEZ is an emerging threat to swine
health. To generate a disease model and evaluate the susceptibility of healthy, conventionally raised pigs to SEZ,
we challenged sows and five-month-old pigs with an isolate from a 2019 mortality event. Pigs were challenged
with a genetically similar guinea pig isolate or genetically distinct horse isolate to evaluate comparative viru­
lence. The swine isolate caused severe systemic disease in challenged pigs with 100 % mortality. Disease
manifestation in sows was similar to field reports: lethargy/depression, fever, reluctance to rise, and high
mortality. The guinea pig isolate also caused severe systemic disease; however, most five-month-old pigs
recovered. In contrast, the horse isolate did not cause disease and was readily cleared from the respiratory tract.
In conclusion, we were able to replicate disease reported in the field. The results indicate differences in virulence
between isolates, with the highest virulence associated with the swine isolate. Additionally, we generated a
challenge model that can be used in future research to evaluate virulence factors and disease prevention
strategies.

1. Introduction 2004; Soedarmanto et al., 1996). The frequency of SEZ detection in

Streptococcus equi subspecies zooepidemicus (SEZ) is a Gram positive
bacterium considered to be a mucosal commensal of horses and most
commonly associated with opportunistic infections in the respiratory or
genital tracts (Timoney, 2004). SEZ is not considered to be host adapted
and it is also a cause of infections in many other mammalian species,
including humans (Baracco, 2019; Timoney, 2004). Zoonotic infection
with SEZ has been linked to animal contact or consumption of animal
products and clinical conditions range from mild disease, such as
pharyngitis, to severe systemic infection (Baracco, 2019).
SEZ has been associated with significant disease in pigs globally.
Historically, outbreaks associated with SEZ have been reported in
Indonesia and China, with mortality during the Chinese outbreak in
1975 estimated around 300,000 animals (Ma et al., 2011; Salasia et al.,
diseased pigs in China has declined in recent years; however, it is still
isolated from animals with streptococcal disease (Wei et al., 2009). In
North America, SEZ infections in swine have been uncommon and the
Iowa State University Veterinary Diagnostic Laboratory reports only six
isolations of SEZ from diagnostic cases from 2009 to 2019 (ISUVDL,
2019; Sitthicharoenchai et al., 2020); however, in 2019, incidents of
high mortality attributed to SEZ in cull sows and feeder pigs were
observed in Ohio, Tennessee, and Manitoba, Canada (Chen et al., 2020;
Costa and Lage, 2020; ISUVDL, 2019; Sitthicharoenchai et al., 2020;
Surendran Nair et al., 2020). Disease was characterized by rapid onset of
severe depression, lethargy, weakness, fever, and mortality levels
ranging from 30 to 50% (Chen et al., 2020; ISUVDL, 2019; Sitthichar­
oenchai et al., 2020). Isolates from the recent mortality events showed
remarkable genetic similarity to each other and a Chinese strain from

* Corresponding author at: 1920 Dayton Ave, Ames, IA, 50010, United States.
E-mail address: susan.brockmeier@usda.gov (S.L. Brockmeier).

https://doi.org/10.1016/j.vetmic.2021.109271
Received 6 April 2021; Accepted 29 October 2021
Available online 2 November 2021
0378-1135/Published by Elsevier B.V.
S.J. Hau et al.
1975 (Chen et al., 2020; Costa and Lage, 2020). The identification of
isolates with high genetic similarity may indicate the importance of this
SEZ isolate as an emerging threat to swine health in North America.
Understanding the susceptibility of pigs to disease and being able to
replicate disease in the laboratory setting is an important step in the
assessment of critical virulence factors and evaluation of disease pre­
vention strategies.
Here, we examined the susceptibility of healthy, conventional cull
sows and five-month-old feeder pigs to the SEZ isolate associated with
the recent swine mortality event in Tennessee. The virulence of the
swine-associated isolate was compared with a genetically distinct horse
isolate and a guinea pig isolate that was genetically clustered with the
swine-associated isolate and harbored many of the same virulence fac­
tors (Chen et al., 2020).
2. Materials and methods
2.1. Bacterial isolates and culture conditions
SEZ isolates were obtained from USDA National Veterinary Services
Laboratories. SEZ 19-029742-19-352-3A (NCBI-SRR10584762, https
://www.ncbi.nlm.nih.gov/sra/SRR10584762) was isolated from the
pleural fluid of a horse with severe pneumonia and a pleural abscess
(Chen et al., 2020). SEZ 19-035701-R130327313 (SRR10512734, https
://www.ncbi.nlm.nih.gov/sra/SRR10512734) was isolated from a
guinea pig lymph node and was associated with human clinical cases
following guinea pig exposure (Chen et al., 2020). SEZ
19-031482-K1916623-LUNG1 (SRR10584760, https://www.ncbi.nlm.
nih.gov/sra/SRR10584760) was isolated from the lung of a sow asso­
ciated with a mortality event in Tennessee (Chen et al., 2020). Isolates
will be referred to throughout by their source: horse, guinea pig, and
swine, respectively.
Isolates were grown on trypticase soy agar with 5% sheep blood
(Becton Dickinson, Franklin Lakes, NJ) or in brain heart infusion broth
(Becton Dickinson) at 37 ◦ C with 5% CO2.
2.2. Sow pathogenesis
All animal studies were approved by the USDA-ARS-National Animal
Disease Center’s Institutional Animal Care and Use Committee. Fifteen
sows were obtained from a commercial sow farm and had farrowed
previously at the National Animal Disease Center (NADC). Sows were
randomly distributed into three groups (n = 5). Challenge inoculum was
grown on blood agar plates and suspended in PBS to an OD600 of
approximately 0.42. Sows were challenged with 5 mL total volume, 1.5
mL administered to each nostril and 2 mL orally. Group 1 sows were
challenged with 7.55 × 108 colony forming units (CFU)/mL of SEZ horse
isolate (numbers: 5455, 5675, 6074, 6159, 6167). Group 2 sows were
challenged with 1.06 × 1010 CFU/mL of SEZ guinea pig isolate
(numbers: 6014, 6340, 6356, 6431, 6788). Group 3 sows were chal­
lenged with 9.15 × 107 CFU/mL of SEZ swine isolate (numbers: 5456,
6004, 6082, 6093, 6808). Nasal and tonsil swabs were taken on days 3
and 7 post-challenge to evaluate colonization in surviving animals. After
challenge, sows were evaluated at least twice daily for clinical signs of
disease. Following development of clinical signs, evaluations were
completed approximately every 4 h excluding an 8 -h overnight period.
Sows were euthanized if clinical signs became severe, such as severe
depression/lethargy, or inability to rise. Clinical signs and gross lesions
were recorded, and the following samples were fixed in formalin and
processed for histopathology: lung, heart, spleen, lymph node, tonsil,
liver, kidney, and small intestine. Samples were taken for bacteriologic
evaluation: nasal swab, tonsil swab, joint fluid, serosal swab, cerebro­
spinal fluid (CSF), bronchoalveolar lavage fluid (BALF), serum, and
splenic swab. Samples were plated on trypticase soy agar with 5% sheep
blood or phenylethyl alcohol agar (Becton Dickinson, Franklin Lakes,
NJ) and incubated at 37 ◦ C with 5% CO2.
2
Veterinary Microbiology 264 (2022) 109271
2.3. Sow re-challenge
Sows surviving challenge with the horse isolate were re-challenged
with the swine isolate 21 days after initial inoculation. Sows were
given 3.3 × 109 CFU/mL of the swine isolate and evaluated daily as
described. Temperature chips (Allflex USA, Inc., Irving, TX) were
applied in the neck muscle of each sow and temperature was monitored
twice daily post-challenge.
2.4. Feeder pig challenge
Eighteen five-month-old pigs raised at the NADC were randomly
distributed into three groups (n = 6). Pigs were challenged with 3 mL of
inoculum [7.9 × 108 (horse), 1.8 × 1010 (guinea pig), and 8.3 × 108
(swine) CFU/mL] given 1 mL per nostril and 1 mL orally. Pigs were
evaluated daily as described and body temperature was monitored twice
daily. At necropsy, samples were taken as described along with a swab of
the kidney, liver and tracheobronchial lymph node. Surviving animals
were euthanized 28 days post challenge.
2.5. Serum chemistry analysis
Serum chemistry panels were completed by Iowa State University
College of Veterinary Medicine’s Clinical Pathology Laboratory on five
of the guinea pig isolate-challenged sows and three of the swine isolate-
challenged sows (6082, 6093, and 6808). Serum obtained at necropsy
was sent for a large animal complete profile which includes concentra­
tions of sodium, potassium, chloride, bicarbonate, calcium, phosphorus,
magnesium, blood urea nitrogen (BUN), creatinine, glucose, total pro­
tein, albumin, aspartate aminotransferase (AST), creatine kinase (CK),
alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and
total bilirubin.
2.6. Systemic cytokine analysis
Systemic cytokine analysis was performed on serum collected at
necropsy using the Cytokine and Chemokine 9-Plex Porcine Procarta­
Plex Panel 1 Kit (Thermo Fisher Scientific) and run on a Luminex
MAGPIX (Luminex Corporation, Austin, TX) as per manufacturers’ rec­
ommendations. Serum levels of interferon (IFN)-α, IFN-γ, interleukin
(IL)-1-β, IL-10, IL-12p40, IL-4, IL-6, IL-8, and tumor necrosis factor
(TNF)-α were measured. Data analysis was completed using the Bio-Plex
Manager MP software (Bio-Rad Laboratories, Hercules, CA).
2.7. S. zooepidemicus antibody response
Serum was collected on day 0 and day 21 or 28 (from surviving
animals) and frozen at -80 ◦ C until ELISAs were performed. SEZ (horse,
guinea pig, and swine) was suspended in PBS to an OD600 of 0.6 and
heat-killed for 30 min at 56 ◦ C. Heat-killed bacteria were diluted 1:10 in
carbonate-bicarbonate buffer and used to coat ELISA plates. Swine
serum was serially diluted and bound antibody was detected with
horseradish peroxidase conjugated secondary antibody specific to swine
immunoglobulin heavy chain (1:20,000 dilution) (SeraCare Life Sci­
ences Inc., Milford, MA) and tetramethylbenzidine substrate (Life
Technologies, Carlsbad, CA). Optical density was measured at 450 nm
with correction at 655 nm and data were modeled using a nonlinear
function of the log10 dilution and the log (agonist)-versus-response
variable slope four-parameter logistic model in GraphPad Prism
(GraphPad Software, San Diego, CA). Endpoint titer was interpolated
using two times the average reading for gnotobiotic swine serum.
2.8. Bacterial growth assessment
Growth rate and peak were assessed using Bioscreen C Automated
Microbiology Growth Curve Analysis System (Growth Curves USA,
S.J. Hau et al.
Piscataway, NJ). Isolates were grown overnight in BHI broth and diluted
to an OD600 = 0.15 in BHI broth. The plate was incubated at 37 ◦ C for 24
h with shaking and OD600 was measured every 20 min. Growth was a
comparison of three biological replicates.
2.9. Capsule evaluation and visualization
Capsule production was evaluated using a hydrophobicity assay
previously described (Rosenberg et al., 1980). Bacterial lawns were
grown on blood agar and suspended in phosphate urea magnesium
sulfate (PUM) buffer to an OD400 of 1.0-1.5. Bacterial suspension (1.2
mL) was placed in a 10 mm round bottom test tube and 0.1 mL xylenes
(Mallinckrodt Pharmaceuticals, Staines-upon-Thames, United Kingdom)
were combined and incubated at 37 ◦ C for 10 min. After vortexing for 2
min, the suspension was rested for 15 min at room temperature. The
aqueous phase was collected and OD400 was measured post-treatment.
Results were calculated as a change in OD400 and used to generate a
percent suspension remaining in solution. Hydrophobic (less encapsu­
lated) material moves into the xylenes phase and highly encapsulated
material remains in the PUM suspension.
Capsule was assessed by transmission electron microscopy (TEM)
using the protocol previously described (Borrathybay et al., 2003; Jac­
ques and Foiry, 1987; Kawamoto et al., 2007), with modifications as
indicated by Eberle et al. (Eberle et al., 2020). Bacteria were suspended
in 0.1 M cacodylate buffer containing 2.5 % glutaraldehyde and 0.1 %
ruthenium red for 2 h. After pelleting, bacteria were resuspended in 0.1
M cacodylate buffer with 2.5 % glutaraldehyde and 1.0 mg/mL of pol­
ycationic ferritin for 30 min. After washing in 0.1 M cacodylate buffer,
samples were post-fixed with 2% osmium tetroxide. Samples were rinsed
in 0.1 M cacodylate buffer, dried, and embedded in Eponate 12 (Ted
Pella Inc., Redding, CA). After polymerization for 48 h, samples were
sectioned and stained with 4% uranyl acetate and Reynolds’ lead stain.
Imaging was completed using a FEI Tecnai G2 BioTWIN electron mi­
croscope (FEI Co., Hillsboro, OR).
2.10. Static biofilm assay
Biofilm production was evaluated using a microtiter plate assay, as
described previously (Cassat et al., 2007). Briefly, overnight cultures of
SEZ were adjusted to an OD600 of approximately 0.42 and diluted 1:2,
1:4, and 1:10 in BHI broth. Diluted SEZ was plated in triplicate in a
96-well flat bottom plate and incubated statically for 48 h at 37 ◦ C with
5% CO2. After 48 h, supernatant was aspirated and the plate was washed
with PBS. The biofilm was fixed with 100 % ethanol, allowed to dry, and
stained with 0.1 % crystal violet for 15 min. After staining, the plate was
washed with PBS and dried overnight. Crystal violet from the biofim
matrix was eluted in 100 % ethanol (150 μL) for 10 min, collected into a
new 96-well plate, and absorbance was measured at 538 nm.
2.11. Serum sensitivity assay
Sensitivity to complement-mediated killing was assessed with guinea
pig serum (GPS) (Quidel, San Diego, CA). SEZ stocks were generated by
suspending plate-grown SEZ in 50 % PBS:glycerol to an OD600 of 0.42
(~1 × 108 CFU/mL) and frozen at -80 ◦ C until use. As a control, GPS was
heat inactivated for 30 min at 56 ◦ C (HI-GPS). In 96-well round bottom
plate, 80 μL of GPS or HI-GPS was combined with 20 μL of SEZ stock
suspensions. After incubation at 37 ◦ C with 100 revolutions per minute
(rpm) shaking for 1 h, serial dilutions were plated for enumeration.
2.12. Phagocytosis susceptibility evaluation
Porcine alveolar macrophages (PAMs) were isolated following pre­
viously described protocols (Cullen and Rycroft, 1994; Hu et al., 2016).
Briefly, healthy swine lungs (n = 4) were flushed with PBS. Aspirated
PBS was centrifuged at 200 x g for 10 min. Pelleted cells were washed
3
Veterinary Microbiology 264 (2022) 109271
and resuspended in complete RPMI-1640 medium (10 % fetal bovine
serum, 1 μg/mL fungizone, 100 U/mL penicillin, and 100 μg/mL
streptomycin). Cells were plated in petri dishes and allowed to adhere
for 2 h at 37 ◦ C with 5% CO2. After incubation, media and non-adherent
cells were aspirated. Adherent cells were washed with complete RPMI
and collected by cell scraping. Cells were pelleted, washed, and resus­
pended in RPMI without antibiotics. Cells were quantified and assessed
for viability with the Countess II Automated Cell Counter (Invitrogen,
Carlsbad, CA) and plated into 48-well plates with 5 × 105 PAMs per well.
After adhering for 20 min, the media was aspirated and media con­
taining diluted, frozen stock of SEZ (approximately 5 × 106 CFU/mL,
MOI 10:1) was added to each well. PAMs and SEZ were incubated for 2 h
at 37 ◦ C with 5% CO2. After incubation, the supernatant was aspirated
and used to quantify non-phagocytosed bacteria. The CFU/mL was
quantified for the inocula and log fold reduction was calculated.
2.13. Adherence and invasion using CCL-30 cells
CCL-30 cells (ATCC, Manassas, VA) were propagated in Eagle’s
Minimum Essential Medium (EMEM) (ATCC). For the assay, CCL-30
cells were plated at 3 × 105 cells/well into 48-well tissue culture
plates. Plates were incubated overnight and washed with PBS prior to
use. Pre-made stocks of SEZ were diluted and cells were inoculated with
0.1 mL of suspension at an MOI of 10:1 (approximately 3 × 106 CFU/
well). Plates were centrifuged at 800 x g for 5 min to settle bacteria. The
bacteria were allowed to adhere to cells for 3 h at 37 ◦ C with 5% CO2.
Following incubation, cell monolayers were washed with PBS. Cells
were lysed with 0.1 mL 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO)
and CFU were enumerated with serial dilutions. Invasion was assessed
by allowing adherence for 2 h, washing with PBS, and incubating with
0.1 mL EMEM containing 25 μg/mL Penicillin G (Sigma Aldrich) for 1 h
at 37 ◦ C with 5% CO2 to kill surface adhered bacteria. The monolayer
was washed with PBS, cells were lysed with 0.1 mL 1% Triton X-100, and
CFU were enumerated. Control wells were treated similarly to test wells
using non-infected EMEM. Cells per well were quantified using a Scepter
Cell Counter (Thremo Fisher Scientific).
2.14. Statistical analysis
Statistical analysis was completed in GraphPad Prism version 8.4.2
(GraphPad, La Jolla, CA). Survival was assessed using the product limit
method of Kaplan and Meier and compared using the log-rank test.
Survival was compared using Fisher’s exact tests. Differences in anti­
body titer, serum cytokine levels, hydrophobicity, biofilm formation,
serum sensitivity, susceptibility to phagocytosis, adherence, and inva­
sion were assessed using one-way ANOVA. A statistical threshold of P <
0.05 was used as a cutoff.
3. Results
3.1. Sow pathogenesis
3.1.1. Clinical signs
Clinical signs for challenged sows are listed in Table S1. Sows chal­
lenged with the horse isolate remained clinically normal throughout the
study period. Most sows challenged with the guinea pig isolate devel­
oped clinical signs 24 h post-inoculation (hpi) (4/5 sows) and the final
sow developed clinical signs 84 hpi, including anorexia and depression.
Sows were euthanized due to the severity of clinical signs at 54 hpi
(6014, 6340), 78 hpi (6356), and 102 hpi (3788) (Fig. 1). The final sow
(6431) recovered and was euthanized 28 days post-inoculation (dpi). All
sows challenged with the swine isolate rapidly developed clinical signs,
with sows going off feed and some episodes of vomiting occurring by 24
hpi. Severe lethargy and depression followed by 36–48 hpi. One sow was
euthanized due to severity of disease at 36 hpi (6082), two were found
dead 48 hpi (5456, 6004), and two were euthanized due to disease
S.J. Hau et al. Veterinary Microbiology 264 (2022) 109271

Fig. 1. Survival of sows after challenge with S. zooepidemicus. None of the
sows challenged with the horse isolate had clinical signs (blue line). Sows
challenged with the guinea pig isolate developed clinical signs rapidly (green
line), but disease progressed more slowly when compared to the swine isolate.
All sows challenged with the swine isolate were euthanized due to disease
severity or succumbed to disease by 54 hpi (red line). There were differences in
median survival time between sows challenged with the guinea pig isolate and
sows challenged with the swine isolate. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of
this article).
severity at 54 hpi (6093, 6808) (Fig. 1).
3.1.2. Gross and histopathologic changes
Sows challenged with the horse isolate were not euthanized and no
assessment of gross or histopathologic changes was completed. Gross
lesions at necropsy were minimal in sows. Two sows challenged with the
guinea pig isolate had gross lesions. One sow (6356) had pancreatic
edema and fat saponification. The sow that recovered from infection
(6431) had small patches of consolidation in the left caudal lung lobe
and fibrous tags on the pleural surface suggesting resolved pleuritis.
Histopathologic lesions in the guinea pig challenged sows were limited.
The most common findings included mild to moderate increased alve­
olar septal neutrophilic infiltration in the lung (5/5) ± interlobar edema
(2/5), mild to moderate hepatic congestion (4/5), fibrous deposits on
the splenic capsule (3/5), and multifocal neutrophilic aggregates in
lymph nodes (2/5). Additionally, one sow had necrosuppurative
lymphadenitis with bacterial colonies and fibrin thrombi (1/5). The
sows challenged with the swine isolate showed mild organ congestion
grossly and the lungs of two sows (6082, 6093) had small areas of
consolidation. The lung of sow 6004 was hemorrhagic and epistaxis was
observed post-mortem. The splenic size was within normal limits for the
sows found dead and consistent with barbiturate euthanasia for eutha­
nized sows. Sows challenged with the swine isolate had histopathologic
lesions consistent with sepsis, including moderate to marked pulmonary
congestion (5/5) and increased alveolar septal neutrophilic infiltrates
(4/5) ± fibrin thrombi and bacterial colonies (1/5), moderate to marked
hepatic congestion (5/5) ± neutrophil infiltration (3/5), splenic
neutrophil aggregates (2/5) or bacteria (1/5), renal congestion with
bacterial colonies in the glomeruli (1/5) and interstitial vessels (2/5),
and necrosuppurative tonsillitis (3/5). Additional cardiac lesions
included epicardial hemorrhage, neutrophil aggregates in the epicar­
dium and pericardial fat, and fibrin thrombi containing bacterial col­
onies (2/5).
3.1.3. SEZ systemic distribution
Systemic distribution of SEZ was evaluated in challenged animals via
bacterial isolation on samples collected at necropsy. The results of SEZ
isolation are presented in Table 1. The sows challenged with the horse
isolate were not assessed for systemic distribution of SEZ. Respiratory
colonization was evaluated on 3 and 7 dpi. Two sows were colonized
with the horse isolate at 3 dpi and all were negative at 7 dpi for SEZ. All
sows inoculated with the guinea pig isolate were colonized with SEZ in
their nasal cavity or tonsil at necropsy. SEZ was also isolated from the
CSF of two sows (6788 and 6431). The other three sows were not sys­
temically positive for SEZ (Table 1). All sows inoculated with the swine
isolate harbored SEZ in the nasal cavity or tonsil at necropsy. Systemic
distribution with the swine isolate varied, with the spleen (4/5) and CSF
(5/5) samples most frequently positive. The sows that died overnight
(5456, 6004) had the greatest CFUs recovered from the systemic samples
and the widest systemic distribution, with all sampled sites positive.
3.1.4. Serum chemistry
Serum chemistry analysis was completed on serum collected at
necropsy for all sows challenged with the guinea pig isolate (5/5) and
three of the sows challenged with the swine isolate (6082, 6093, 6808).
Serum chemistry abnormalities are listed in Table S2. At necropsy
challenged sows showed evidence of dehydration (elevated total protein
and/or albumin), muscle damage (elevated creatine kinase), and po­
tential hepatocellular or muscle damage (elevated aspartate amino­
transferase) The sow that recovered following challenge with the guinea
pig isolate had no notable serum chemistry abnormalities.

Table 1
Systemic distribution of S. zooepidemicus in sows at necropsy.
Nasala Tonsil Jointb Serosa CSF BALF Serum Spleen

Challenged with Guinea Pig Isolate

6014 + + 0 0 0 0 0 0
6340 + – 0 0 0 0 0 0
6356 + – 0 0 0 0 0 0
6788 – + 0 0 + 0 0 0
6431 – + 0 0 + 0 0 0

Challenged with Swine Isolate

6082 + – 0 + + + ++ +++
5456 + – +++ +++ +++ +++ +++ +++
6004 + + ++ +++ +++ +++ +++ +++
6093 – + 0 0 + 0 0 0
6808 + – 0 0 + 0 0 +++

Re-Challenged with Swine Isolate

6074 + + 0 ++ 0 0 0 ++
5455 + – 0 +++ 0 0 0 ++
6159 + + 0 0 0 0 0 0
5675 + + 0 +++ 0 0 0 +
6167 + – 0 ++ 0 0 0 0
a
Nasal and tonsil swabs were not plated for enumeration, but streaked for isolation to detect S. zooepidemicus presence (+) or absence (–).
b
Joint, serosa, CSF, BALF, serum, and spleen were plated for enumeration with 100 μL spread on each plate; numbers represent CFU/100 μL with quantification as
follows: 0–10 colonies (+), 10–100 colonies (++), and greater than 100 colonies (+++).

4
S.J. Hau et al.
3.1.5. Systemic cytokines
The cytokine response of sows at necropsy was compared to that of
sows prior to challenge (D0). Though statistical significance was not
reached, the swine isolate challenged sows tended to have cytokine
levels that differed from the pre-challenge group, while the guinea pig
challenged sows tended to have cytokine levels similar to that of the pre-
challenge group (Fig. S1-A). Specifically, IFN-α, IL-10, IL-12p40, IL-6,
and IL-8 were elevated and IL-1-β and IL-4 were reduced in sows chal­
lenged with the swine isolate when compared with D0 samples.
3.2. Sow re-challenge
To assess the impact of prior SEZ exposure on development of SEZ
disease with the swine isolate, sows surviving challenge with the horse
isolate were re-challenged with the swine isolate after 21 days. Two
sows were found to be colonized with the SEZ horse isolate at the time of
re-challenge.
3.2.1. Clinical signs
Clinical signs developed in 2/5 sows 24 hpi, 2/5 sows 48 hpi, and 1/5
sows 96 hpi. Sows developed similar clinical signs to that of primary
challenge (Table S1) and fever was observed in all challenged animals
(Fig. 2-A). Additionally, sow 6167 developed an enlarged, reddened
vulva and purulent vulvar discharge. Sows were euthanized 2 dpi
(6074), 3 dpi (5455), 4 dpi (6159), 6 dpi (5675), and 10 dpi (6167) due
to worsening clinical disease (Fig. 2-B).
3.2.2. Gross and histopathologic changes
More gross lesions were noted in the re-challenged sows than sows
receiving primary challenge with the swine isolate. Lesions included
pleuritis (4/5), peritonitis (3/5), and lung consolidation or pulmonary
edema (3/5). One sow had an enlarged spleen and two had pancreatic
lesions similar to that seen in the sow challenged with the guinea pig
isolate. Sow 6167 had gross reproductive tract lesions including
oophoritis and salpingitis. Re-challenged sows developed histologic le­
sions consistent with sepsis. Lesions included mild to moderate
increased alveolar septal pulmonary neutrophilic infiltrates (5/5) ±
interlobular edema (3/5) and pulmonary congestion (2/5), moderate to
marked hepatic congestion (2/5), mild hepatic neutrophil infiltrates (2/
5), neutrophil infiltration and sinusoid edema in lymph nodes (3/5), and
fibrinosuppurative peritonitis (3/5). Less common lesions included
necrosuppurative alveolitis and fibrinosuppurative pleuritis (1/5), sup­
purative and hemorrhagic lymphadenitis (1/5), necrotizing steatitis and
saponification of the peripancreatic fat (1/5), and neutrophils and
necrotic debris on the ovarian germinal epithelium (1/5).
3.2.3. SEZ systemic distribution
SEZ distribution is listed in Table 1. SEZ was not as widely distrib­
uted in re-challenged sows as was seen in primary challenged sows.
Culture results coincided with lesions and the serosal swab was the most
isolate were compared (B). All sows that received a primary challenge with the S. zooepidemicus swine isolate were euthanized due to severity of disease by 54 hpi
(red line). Sows that received the horse isolate as the primary challenge and were re-challenged with the swine isolate (blue line) had a longer median survival time,
with the first sow being euthanized due to worsening disease at 54 hpi and the final sow being euthanized 10 days post infection. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article).
5
Veterinary Microbiology 264 (2022) 109271
frequently positive sample (4/5).
3.2.4. Serum chemistry
Serum chemistry analysis on the re-challenged sows revealed
changes similar to primary challenged sows and are listed in Table S2.
The re-challenged sows did not develop the electrolyte changes seen in
primary challenged sows.
3.2.5. Systemic cytokine assessment
Systemic cytokine levels in re-challenged sows were compared to
sows on D0 and the sows that received primary challenge with the swine
isolate (Fig. S1-B). The cytokine levels in re-challenged sows were more
consistent with D0 samples than the primary challenged sows. Re­
ductions in IL-1-β and IL-4 were changes consistent between sows
receiving primary challenge and re-challenged sows.
3.2.6. Serum antibody response
The serum antibody titers to the SEZ horse isolate and swine isolate
were quantified for the sows pre-challenge (D0) and on day 21 post
challenge with the horse isolate (Fig. S2). The antibody titers were
comparable between the swine and horse isolate at the time of chal­
lenge. There were also no statistical differences in D0 and D21 titers;
however, a numerical rise in titer was observed for both the horse and
swine isolate.
3.3. Feeder pig challenge
3.3.1. Clinical signs
Clinical signs following challenge of feeder pigs are listed in
Table S3. Feeder pigs challenged with the horse isolate did not show any
clinical signs of disease and none were febrile (Fig. 3-A). Feeder pigs
challenged with the guinea pig isolate developed clinical signs similar to
that seen in sows by 24 hpi including elevated temperature (Fig. 3-A).
The guinea pig isolate caused severe clinical signs necessitating eutha­
nasia in 2 pigs with neurologic signs (paddling or inability to rise)
developing 5 and 6 dpi (Fig. 3-B). The pigs challenged with the guinea
pig isolate remained depressed and lethargic for approximately 9 dpi,
but they were less severely affected than feeder pigs challenged with the
swine isolate and 4/6 recovered. Feeder pigs challenged with the swine
isolate developed fever (Fig. 3-A) and other clinical signs 24 hpi. The
pigs succumbed to disease or were euthanized due to the severity of
clinical signs at 48 hpi (1/6) and 72 hpi (5/6) (Fig. 3-B).
3.3.2. Gross and histopathologic changes
The feeder pigs challenged with the horse isolate were predomi­
nantly free of gross lesions at necropsy 28 dpi. One pig had small patches
of consolidation in the left cranial lung lobe. One of the two pigs
euthanized due to severity of disease with the guinea pig isolate had
pleuritis, peritonitis, and renal petechiae and the other had no signifi­
cant lesions at necropsy. Only one pig recovering from challenge with
Fig. 2. Temperature response and survival
of sows re-challenged with the
S. zooepidemicus swine isolate. The tempera­
ture of sows following re-challenge with the
S. zooepidemicus swine isolate was tracked (A).
Normal sow body temperature is indicated by
the dashed line. Each sow is plotted individu­
ally. The sows rapidly developed an elevated
temperature with all sows having a temperature
≥40.0 ◦ C at multiple time points following
challenge. Survival following challenge and re-
challenge with the S. zooepidemicus swine
S.J. Hau et al.
the guinea pig isolate had gross lesions at necropsy 28 dpi, which were
small areas of consolidation in the right middle and cranial and left
middle lung lobes. Feeder pigs challenged with the swine isolate
developed more gross lesions than sows receiving primary challenge
with the swine isolate. Lesions included peritonitis (4/6), pulmonary
consolidation (1/6), pulmonary hemorrhage (2/6), dark spleen with
pale margins (2/6), hemorrhagic lymph nodes (1/6), renal petechia (1/
6), and dark kidneys (3/6). Histologic lesions after challenge with the
horse isolate were minimal and included mild to moderate increased
alveolar septal pulmonary neutrophilic infiltrates (6/6). One animal
challenged with the horse isolate had granulomatous pneumonia with
the presence of foreign material, consistent with aspiration pneumonia.
Challenge with the guinea pig isolate resulted in more histologic lesions.
Animals that were euthanized in the acute phase of disease (2/6) had
marked neutrophilic infiltrates in alveolar septa (2/2) ± interlobular
septal edema (1/2), hepatic sinusoid congestion with moderate
neutrophil infiltration (2/2), splenic neutrophil infiltration (1/2), and
edema in the lymph node sinusoids (1/2). Pigs that recovered from
challenge with the guinea pig isolate had lesions including moderate to
marked neutrophilic infiltrates in alveolar septa (4/4) ± interlobular
septal edema (2/4) and fibrin thrombi (1/4), and neutrophil infiltration
and congestion in the lymph node sinusoids (3/4). Lesions in pigs
challenged with the swine isolate included moderate to marked
neutrophilic infiltrates in alveolar septa (6/6), marked pulmonary
congestion (4/6) ± interlobular edema (3/6) and pulmonary hemor­
rhage (3/6), moderate to marked hepatic congestion (5/6) and neutro­
phil infiltration (6/6), splenic neutrophil infiltration (3/6), lymph node
sinusoid congestion (4/6) ± neutrophil infiltration (3/6), fibrin depo­
sition (2/6) and hemorrhage (2/6), and marked renal vascular conges­
tion (4/6). One swine isolate-challenged animal also had necrotizing
tonsillitis with epithelial ulceration and streaming leukocytes.
6
Veterinary Microbiology 264 (2022) 109271
Fig. 3. Feeder pig temperature and survival following
S. zooepidemicus challenge. Individual animal temperatures
post challenge are plotted (A) and fever is indicated by the
dashed line (40 ◦ C). All animals challenged with the guinea pig
isolate (green lines) and swine isolate (red lines) rapidly
developed a fever and were febrile at one or more time points
post-challenge. None of the animals challenged with the horse
isolate (blue lines) developed a fever post challenge. Survival
following feeder pig challenge indicated differential survival
between the swine isolate and the horse and guinea pig isolates
(B). The swine isolate was the most virulent causing 100 %
mortality. Challenge with the guinea pig isolate resulted in 33
% mortality and the horse isolate cause no clinical signs of
disease (0% mortality). There was no statistical difference in
survival between the pigs challenged with the horse and guinea
pig isolates. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of
this article).
3.3.3. SEZ systemic distribution
The systemic distribution of SEZ in feeder aged pigs is listed in
Table 2. Feeder pigs challenged with the horse isolate were necropsied
and sampled 28 dpi. No SEZ was isolated from the systemic sites of the
pigs challenged with the horse isolate. SEZ was not isolated from the
nasal cavity of any pigs; however, two pigs harbored SEZ in their tonsil
at necropsy. Most of the feeder pigs challenged with the guinea pig
isolate (4/6) recovered and were euthanized at 28 dpi. In the two ani­
mals euthanized due to disease progression (315 and 318), the only
systemic site positive was the CSF sample. Most of the feeder pigs sur­
viving challenge with the guinea pig isolate (3/4) were systemically
negative for SEZ; however, the lymph node of one pig was positive for 28
dpi at necropsy. Additionally, 28 dpi in pigs that recovered from chal­
lenge with the guinea pig isolate, SEZ was found in the tonsil of 2/4 but
not detected in any of the nasal samples. All swine isolate challenged
feeder pigs were euthanized due to disease severity and SEZ was found
at multiple systemic sites. The most common isolation site in feeder aged
pigs was the CSF (6/6), liver (6/6), and spleen (5/6), with CSF having
the highest concentration of bacteria in most cases (Table 2).
3.3.4. Systemic cytokine assessment
The systemic cytokine levels of feeder pigs that were euthanized
during the acute phase of disease (guinea pig isolate challenge [n = 2]
and swine isolate challenge [n = 6]) were evaluated and compared with
cytokine levels from feeder pigs prior to challenge (D0) (Fig. S1-C). The
differences did not meet the statistical threshold; however, numerical
changes similar to challenged sows were noted. Animals challenged with
the guinea pig isolate had cytokine levels similar to animals pre-
challenge. Animals challenged with the swine isolate had changes in
eight of the nine evaluated cytokines. Elevations in IFN-α, IL-1-β, IL-10,
IL-12p40, IL-6, IL-8, and TNF-α and a reduction in IFN-γ were seen at
necropsy in pigs challenged with the swine isolate.
S.J. Hau et al. Veterinary Microbiology 264 (2022) 109271

Table 2
Systemic distribution of S. zooepidemicus in feeder pigs at necropsy.
Nasala Tonsil Jointb Serosa CSF BALF Serum Spleen Liver Kidney LN

Challenged with Horse Isolate

301 – – 0 0 0 0 0 0 0 0 0
303 – – 0 0 0 0 0 0 0 0 0
304 – + 0 0 0 0 0 0 0 0 0
305 – – 0 0 0 0 0 0 0 0 0
306 – – 0 0 0 0 0 0 0 0 0
309 – + 0 0 0 0 0 0 0 0 0

Challenged with Guinea Pig Isolate

310 – + 0 0 0 0 0 0 0 0 0
315 – – 0 0 1 0 0 0 0 0 0
316 – – 0 0 0 0 0 0 0 0 0
318 þ – 0 0 TNTC 0 0 0 0 0 0
319 – – 0 0 0 0 0 0 0 0 0
320 – + 0 0 0 0 0 0 0 0 1

Challenged with Swine Isolate

321 – − 28 TNTC 134 95 TNTC TNTC TNTC TNTC TNTC
323 – + 0 0 TNTC 0 0 36 250 0 28
324 – + 0 0 TNTC TNTC 0 0 TNTC 6 7
326 þ – 0 4 TNTC TNTC 27 102 TNTC 5 12
327 þ + 0 TNTC TNTC TNTC 3 33 250 7 6
329 – – 0 0 TNTC 0 0 18 10 0 0
a
Nasal and tonsil swabs were not plated for enumeration, but streaked for isolation to detect S. zooepidemicus presence (+) or absence (–).
b
Joint, serosa, CSF, BALF, serum, and spleen were plated for enumeration with 100 μL spread on each plate; numbers represent CFU/100 μL; TNTC represents “too
numerous to count”.

3.3.5. Serum antibody response
Antibody specific to SEZ was quantified for feeder pigs prior to
challenge (D0) and 28 dpi (D28) for the pigs surviving challenge with
the horse isolate and recovering from challenge with the guinea pig
isolate (Fig. S3). Increased titers were seen at 28 dpi for pigs challenged
with both the horse and guinea pig isolates. The antibody titers to all SEZ
strains were numerically higher 28 dpi for pigs challenged with the
guinea pig isolate; however, the titer to the swine isolate was statistically
higher 28 dpi in animals challenged with the guinea pig isolate than
animals challenged with the horse isolate (P = 0.03).

Fig. 4. Extracellular polysaccharide assessment and bio­

film formation. Production of extracellular polysaccharide
was quantified using a hydrophobicity assay (A) and visually
assessed with transmission electron microscopy (B–D). There
were no differences in the hydrophobicity assessment with all
isolates having around 35 % of the initial suspension remaining
after removal of hydrophobic components. When isolated col­
onies were harvested, the thickness of the polysaccharide on
the cell surface was similar between the horse (B) and guinea
pig (C) isolates; however, the swine (D) isolate was more
heavily encapsulated (E). Biofilm production was assessed
using a static biofilm assay (F). No statistical differences were
noted between the isolates, though the guinea pig isolate
consistently produced more biofilm than the swine or horse
isolate.

7
S.J. Hau et al.
3.4. Bacterial growth assessment
To detect differences in growth rate that may have contributed to the
increased virulence of the swine and guinea pig isolates, growth rate and
peak were compared for the isolates. The growth rate of the guinea pig
and horse isolate were similar; however, the swine isolate grew more
slowly than the other isolates. Additionally, the growth peak for the
swine isolate was lower than that of the guinea pig and horse isolate
(Fig. S4).
3.5. Colony morphology, capsule production, and biofilm formation
Capsule is known to play a major role in the virulence of systemic
bacterial pathogens of swine (Charland et al., 1998; Eberle et al., 2020;
Wang et al., 2013). For the SEZ isolates utilized in this study, isolated
colonies of the swine isolate were visibly more mucoid when grown on
agar media as compared with the horse and guinea pig isolates. To assess
production of capsular polysaccharide, the isolates were evaluated uti­
lizing a hydrophobicity assay and TEM to quantify and visualize extra­
cellular polysaccharide of agar grown bacterial lawns. Isolates were
found to have high hydrophobicity with only 36–38 % of the initial
suspension being retained in the aqueous phase (Fig. 4). There was no
statistical difference noted between the horse, guinea pig, and swine
isolates (P = 0.70) in hydrophobicity and therefore no difference in
extracellular polysaccharide production. TEM revealed no difference in
surface polysaccharide thickness between the horse, guinea pig, and
swine isolates when grown as lawns or in broth (data not shown);
however, when isolated colonies were evaluated, the swine isolate was
more heavily encapsulated than the horse or guinea pig isolate
(Fig. 4-B-D). Biofilm production is thought to contribute to colonization
of the nasal cavity and development of systemic disease with strepto­
coccal species (Blanchette-Cain et al., 2013; Marks et al., 2014). Biofilm
formation of the SEZ isolates used in this study was evaluated and no
statistical differences were found between the three isolates (Fig. 4-E).
3.6. In vitro assessment of host-pathogen interaction
Differences in host-pathogen interactions are important contributors
to differences in the virulence of bacterial strains. Because of this, the
interaction between SEZ and the innate immune system as well as the
capacity of SEZ to adhere and invade epithelial cells was assessed.
Sensitivity to the innate immune system was evaluated using a serum
sensitivity assay and determining susceptibility to phagocytosis by
porcine alveolar macrophages (PAMs). Serum sensitivity was evaluated
utilizing guinea pig serum (GPS) as a source of complement. None of the
isolates showed sensitivity to complement mediated lysis and there were
no differences in susceptibility between the isolates (Fig. S5-A). Addi­
tionally, none of the isolates showed susceptibility to phagocytosis by
PAMs and all isolates replicated in the cell culture medium over the
incubation period (Fig. S5-B).
The adherence and invasion capacity of the SEZ isolates in this study
was tested using a human squamous cell carcinoma line (CCL-30)
(Fig. S5-C and S5-D). The horse isolate adhered significantly better than
the guinea pig and swine isolates to CCL-30 cells (P < 0.01). The guinea
pig and swine isolates adhered similarly. Invasion of CCL-30 cells was
statistically similar for all isolates, though the horse isolate invaded at
higher numbers than the guinea pig or swine isolate (P = 0.1059 and
0.1241 for the guinea pig and swine isolates, respectively).
4. Discussion
Historically, SEZ has been associated with severe mortality in pigs in
China and Indonesia (Ma et al., 2011; Soedarmanto et al., 1996). More
recently, isolates with high genetic similarity have caused severe mor­
tality events in the U.S. and Canada (Chen et al., 2020; Costa and Lage,
2020; ISUVDL, 2019; Sitthicharoenchai et al., 2020; Surendran Nair
8
Veterinary Microbiology 264 (2022) 109271
et al., 2020). To better understand SEZ virulence and develop a disease
model to assess pathogenesis and determine intervention strategies, we
challenged sows and feeder pigs with three SEZ isolates obtained from
different host species (horse, guinea pig, and swine).
Here, we saw that oronasal challenge of healthy, non-stressed sows
with the swine isolate resulted in severe systemic disease and fatalities
similar to what was reported during the mortality events in Tennessee
and Ohio, with 100 % mortality occurring by 3 dpi. Consistent with case
reports, gross lesions were minimal in naïve challenged sows and his­
tologic lesions were consistent with bacterial septicemia (Sitthichar­
oenchai et al., 2020). The systemic distribution and bacterial load of SEZ
in animals that succumbed to disease was greater than that seen in an­
imals euthanized before end stage disease. This may be due to prolif­
eration and dissemination in the peri-mortem or post-mortem period.
We noted trends in the serum cytokine levels of sows challenged with
the swine isolate that indicated potential dysregulation of cytokines
during infection. Individual animal variation, small group size, and
timing of sampling contributed to the cytokine differences not meeting
the statistical threshold; however, future studies should investigate
cytokine levels, as dysregulation may be playing a role in the rapid
mortality associated with infection.
We also challenged sows with two non-swine SEZ isolates to compare
virulence in the isolates. The genetically distinct horse isolate, which
lacked the genomic islands and many virulence factors found in the
swine outbreak isolates (Chen et al., 2020), did not cause disease in
inoculated sows. Additionally, the isolate did not colonize the nasal
cavity or tonsil well, as only intermittent carriage was noted
post-challenge. The guinea pig isolate, with high genetic relatedness to
the swine isolate including similar genomic islands and a similar com­
plement of virulence genes (Chen et al., 2020), caused severe systemic
disease in sows that mirrored infection with the swine isolate. While
there was no statistical difference in survival between sows challenged
with the guinea pig and the swine isolates, the median survival time was
longer for sows challenged with the guinea pig isolate and euthanasia
was elective due to worsening condition in all sows challenged with the
guinea pig isolate as compared to 3/5 with the swine isolate. Histolog­
ically, the guinea pig isolate caused similar but milder lesions than those
seen with the swine isolate and no intralesional bacteria were observed.
We also found limited systemic distribution of the guinea pig isolate as
compared with the swine isolate, with the guinea pig isolate recovered
only from the CSF of 2/5 animals, while the swine isolate was recovered
from the CSF of 5/5 animals and from multiple sites in 4/5 animals.
Sows surviving challenge with the horse isolate were re-challenged
with the swine isolate 21 days later. Previous exposure to SEZ did not
provide protection for sows re-challenged with the swine isolate. Sows
rapidly developed clinical signs of disease and were euthanized by 10
dpi. There was no difference in survival between previously exposed
sows and sows with no prior SEZ exposure; however, the median sur­
vival time was lengthened in sows with prior exposure to SEZ (P =
0.014). This prolonged survival may have contributed to increased gross
lesions seen in pre-exposed sows as compared to sows without prior
exposure to SEZ. Additionally, SEZ was less widely disseminated and
isolation correlated with the lesions seen (pleuritis and peritonitis).
While it did not provide protection from disease, there was a small,
numerical rise in titer to both the horse and swine SEZ isolates in sows
exposed to the horse isolate. The increase was larger for the horse isolate
than the swine isolate, indicating only partial cross reactivity between
isolates. The rise in titer may have contributed to prolonged survival
seen in sows previously exposed to SEZ.
Challenge of feeder aged pigs provided better insight into the dif­
ferences in severity of disease and virulence between the evaluated
isolates. Disease severity in feeder pigs challenged with the guinea pig
isolate was reduced as compared with sows. Though there was no sta­
tistical difference in survival, the majority of feeder pigs challenged with
the guinea pig isolate survived challenge (4/6), while the majority of
sows were euthanized (4/5). Differences in virulence were more
S.J. Hau et al.
pronounced between the guinea pig and swine isolates in feeder pigs,
with the swine isolate causing significantly higher mortality than the
guinea pig isolate in this age group. Additionally, while feeder pigs
challenged with the guinea pig isolate showed similar clinical signs and
prevalence of disease, the clinical signs associated with the swine isolate
were more severe. Similar changes in systemic cytokines were noted in
feeder pigs and sows challenged with the swine, though the statistical
threshold was not met in either group. As was noted in sows, the guinea
pig isolate was not widely distributed systemically in the feeder pigs and
was only found in the CSF of pigs that were euthanized due to disease
severity. The swine isolate was widely distributed in feeder pigs as was
seen in sows, with all systemic sites evaluated being positive in one or
more animals. The wide dissemination of the swine isolate in both sows
and feeder pigs could indicate adaption of this isolate to the swine host.
The immune response to challenge was evaluated by assessing
antibody titers, which we compared both between the day of challenge
(D0) and 21 dpi or 28 dpi and between sows and feeder pigs, respec­
tively. A rise in titer was seen over the study period for pigs surviving
challenge. This rise was not statistically significant in sows; however, the
rise in was statistically significant in feeder pigs surviving challenge
with the horse isolate or recovering from challenge with the guinea pig
isolate. Higher titers to the swine isolate were generated by challenge
with the guinea pig isolate than challenge with the horse (P = 0.03). This
may be associated with the genetic similarity between the swine and
guinea pig isolates (Chen et al., 2020). It could also be due to greater
immune stimulation following challenge with the guinea pig isolate, as
animals challenged with the guinea pig isolate developed clinical dis­
ease, while animals challenged with the horse isolate remained clini­
cally normal. Interestingly, sows had significantly higher titers to all
isolates on D0 than the feeder pigs (P < 0.01). This may be associated
with commensal SEZ exposure; however, increased SEZ surveillance has
not resulted in high rates of isolation from domestic swine (ISUVDL
unpublished data). The increased titers in older animals could also be
due to exposure to other bacterial species having epitopes that cross
react with SEZ, though the higher titers did not provide protection from
disease in challenged sows.
The isolates used in this study were evaluated for characteristics
often associated with virulence, such as capsule production, biofilm
formation, serum sensitivity, susceptibility to phagocytosis, and adher­
ence and invasion. There were no differences in biofilm formation,
serum sensitivity, and susceptibility to phagocytosis between the chal­
lenge isolates; however, differences in capsule production were seen
based on culture conditions. The hydrophobicity of SEZ isolates har­
vested from lawns was consistent with a capsule deficient Streptococcus
suis isolate (Faulds-Pain and Wren, 2013) (data not shown), indicating
lawn grown SEZ is not highly encapsulated; however, when isolated
colonies of the swine isolate were compared with lawn growth via TEM,
a large differences was noted in the thickness of the capsule, which could
contribute to the virulence of this isolate in vivo. The differences in
capsule production noted in the swine isolate are consistent with pre­
vious reports indicating SEZ capsule production can be unstable during
laboratory culture (Timoney, 2004). The isolates used in this study
showed minimal susceptibility to complement mediated killing and
were not readily phagocytosed by PAMs, characteristics which are often
associated with encapsulation; however, in this case other virulence
genes, such as the M-like protein, may be facilitating this phenotype. The
horse isolate adhered better to CCL-30 cells in vitro than the guinea pig
or swine isolate. Previously, the capacity for adherence and invasion of
SEZ has been negatively correlated with encapsulation (Xu et al., 2016);
however, we found no differences in the production of capsule between
the three isolates under the culture conditions used for these assays. In
this case, there may be differences in the genetic capacity for adherence,
as the horse isolate was not found to cluster phylogenetically with the
guinea pig and swine isolates (Chen et al., 2020), which were more
similar in their capacity for adherence and invasion.
In the study reported here, we successfully replicated disease with a
9
Veterinary Microbiology 264 (2022) 109271
swine isolate of SEZ that was comparable to reports during high mor­
tality events in North America in 2019 (Costa and Lage, 2020; ISUVDL,
2019; Sitthicharoenchai et al., 2020; Surendran Nair et al., 2020). This
new model will enable future evaluation of virulence factors and
assessment of intervention strategies. We also compared the swine
isolate from a high mortality event to two SEZ isolates from other host
species, a closely related guinea pig isolate and distantly related horse
isolate, finding difference in virulence in the swine host that did not
correlate completely with the previously identified genomic island or
virulence factor complement (Chen et al., 2020). While encapsulation
may be playing a role in the elevated virulence of the swine isolate in
vivo, further investigation into the genetic differences and potential
virulence genes may reveal additional factors that contribute to the
differences in virulence of the swine and guinea pig isolates.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors would like to thank Steven Kellner and Ashley Winter­
owd for their laboratory assistance. The authors also thank the animal
care staff for their assistance in handling and observation of the animals.
Mention of trade names or commercial products in this article is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the USDA. USDA is an equal
opportunity provider and employer. Funding sources had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.vetmic.2021.109271.
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