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Cellular Microbiology of Mycoplasma canis

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DOI: 10.1128/IAI.01440-15

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Cellular Microbiology of Mycoplasma canis

Dina L. Michaels,a Jeffrey A. Leibowitz,a Mohammed T. Azaiza,a Pollob K. Shil,a Suzanne M. Shama,a Gerald F. Kutish,b
Steven L. Distelhorst,c Mitchell F. Balish,c Meghan A. May,d Daniel R. Browna
Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USAa; Department of Pathobiology and
Veterinary Science and Center of Excellence for Vaccine Research, University of Connecticut, Storrs, Connecticut, USAb; Department of Microbiology, Miami University,
Oxford, Ohio, USAc; Department of Biomedical Sciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine, USAd

Mycoplasma canis can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The
unexpected presence of M. canis in brains of dogs with idiopathic meningoencephalitis prompted new in vitro studies to help fill
the void of basic knowledge about the organism’s candidate virulence factors, the host responses that it elicits, and its potential
roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P < 0.01) among strains of M. ca-
nis isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82
histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protec-
tion assays and three-dimensional immunofluorescence imaging. Strains further varied (P < 0.01) in the extents to which they
influenced the secretion of tumor necrosis factor alpha (TNF-␣) and the neuroendocrine regulatory peptide endothelin-1 by
DH82 cells. Inoculation with M. canis also decreased major histocompatibility complex class II (MHC-II) antigen expression by
DH82 cells (P < 0.01), while secretion of gamma interferon (IFN-␥), interleukin-6 (IL-6), interleukin-10 (IL-10), and comple-
ment factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome
sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of M. canis polyvalent antigen distri-
bution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen, M.
canis has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemi-
cal in vivo milieu.

M ycoplasma canis infects many mammalian hosts but is usu-

ally thought of as a commensal or opportunistic cofactor in
respiratory or urogenital tract diseases of dogs (1). We found un-
Congresses of the International Organization for Mycoplas-
mology [90, 91].)

expectedly that M. canis was also detectable by culture or PCR in a
majority of brain tissue specimens in a retrospective case-control
study of canine granulomatous meningoencephalitis (ME)
(GME) and necrotizing ME (NME) (2). The presence of M. canis
in brain tissue was associated with both GME and NME (both P ⬍
0.05, as determined by a ␹2 test). The clinical signs of this common
idiopathic neurological disease of dogs include seizures, proprio-
ceptive deficits, circling, and blindness. Immunosuppressive ther-
apy may be palliative, but the syndrome is progressive and uni-
formly fatal (3). The extensive search for a presumed viral cause of
canine GME and NME has been fruitless (4).
In humans, bacterial meningitis and encephalitis are multifac-
torial lethal infections with often severe sequelae for survivors.
New detection methods have shown that the variety of bacteria
associated with human ME is much more extensive than usually
appreciated (5–9). Additional animal models of bacterial ME are
necessary to study this broader spectrum of pathogens (10). Since
a possible association between M. canis and canine ME was dis-
covered, our objective has been to help fill the void of basic knowl-
edge about the organism’s virulence factors, the host responses
that it elicits, and its potential roles in pathogenesis. Our working
hypotheses were that M. canis is capable of evoking host cell re-
sponses that favor dissemination from mucosal surfaces to sec-
ondary sites of infection, possibly in a strain-dependent fashion,
and also that, regardless of how it might reach those sites, the
presence of M. canis there modulates inflammation and direct
injury to host cells. Understanding this potential can be expected
to help evaluate the cause of canine ME and other diseases.
(Portions of these data were presented in abstract form at
MATERIALS AND METHODS
Mycoplasma strains and cultivation. Strain PG14T of M. canis (ATCC
19525) was first isolated from the throat of a normal dog (11). Strains
UF31, UF33, LV, 5, 26, Cal, and Mara were first isolated from vaginal
swabs of dogs without ME (12). Strains UFG1, UFG2, UFG3, and UFG4
were isolated from frozen brain tissues from cases of canine NME (2).
Mycoplasma cynos strain H-831T (ATCC 27544) was first isolated from the
lung of a dog with pneumonia (13). Mycoplasma arginini strain G230T,
Mycoplasma bovigenitalium strain PG11T, Mycoplasma edwardii strain
PG24T, Mycoplasma maculosum strain Skotti B, Mycoplasma molare strain
H542T, Mycoplasma opalescens strain MH5408T, and Mycoplasma spu-
mans strain PG13T, representing other species that have been isolated
from dogs (1), were obtained from The Mollicutes Collection. All strains
were propagated under standard conditions (14) in ATCC 988 medium
supplemented with fetal bovine serum (FBS) and glucose or arginine.
Stock culture density expressed in CFU was determined by serial dilution
and colony counting after 5 to 7 days of incubation.
Received 24 November 2015 Returned for modification 28 December 2015
Accepted 28 March 2016
Accepted manuscript posted online 4 April 2016
Citation Michaels DL, Leibowitz JA, Azaiza MT, Shil PK, Shama SM, Kutish GF,
Distelhorst SL, Balish MF, May MA, Brown DR. 2016. Cellular microbiology of
Mycoplasma canis. Infect Immun 84:1785–1795. doi:10.1128/IAI.01440-15.
Editor: C. R. Roy, Yale University School of Medicine
Address correspondence to Daniel R. Brown, drbrown@ufl.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/IAI.01440-15.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

June 2016 Volume 84 Number 6 Infection and Immunity iai.asm.org 1785

Michaels et al.
Scanning electron microscopy. M. canis strain PG14T cells were pre-
pared for scanning electron microscopy (SEM) as previously described
(15), with minor modifications. Briefly, glass coverslips were placed into
wells of a 24-well plate. In each well, 100 ␮l of an M. canis stock was
inoculated into 400 ␮l SP-4 broth supplemented with 3% gelatin. After 3
h at 37°C, coverslips were fixed for 30 min at room temperature in 1.5%
glutaraldehyde–1% paraformaldehyde– 0.1 M sodium cacodylate (pH
7.2), rinsed with 0.1 M sodium cacodylate (pH 7.2) five times for 10 min,
and dehydrated through a series of ethanol washes from 25% to 100%
ethanol. The coverslips were then critical-point dried and gold coated.
Images were viewed on a Zeiss Supra 35 (full-frame E-mount, Gold series)
variable-pressure scanning electron microscope operating at 4 kV.
Anti-M. canis polyclonal antibody production, purification, and
validation. Polyclonal antisera against whole-cell lysates (16) of M. canis
strains PG14T, UF33, and UFG1 were generated individually in rabbits.
Antiserum production was performed by Lampire Biological Laboratories
with approval of its Institutional Animal Care and Use Committee. The
protocol consisted of initial subcutaneous inoculation with lysate plus
complete Freund’s adjuvant, followed by boosters of lysate plus incom-
plete Freund’s adjuvant. Seroconversion and strain cross-reactivity were
assessed by standard indirect enzyme-linked immunosorbent assay
(ELISA) methods. All nine homologous and heterologous antigen-antise-
rum combinations were examined.
Total IgG was then purified from each antiserum by circulation
through Sepharose-coupled protein G affinity chromatography columns
according to procedures recommended by the supplier (catalog no. 17-
0618-05; GE Healthcare). For each antiserum, eluate fractions with peak
A280 values were pooled, dialyzed against 1⫻ phosphate-buffered saline
(PBS), and then desalted and concentrated to a final protein concentra-
tion of 15 mg/ml. A portion of each preparation was labeled with biotin
according to procedures recommended by the supplier (catalog no.
PI21329, PI89889, and PI28005; Pierce) for use as immunohistochemical
probes. The working dilutions of the biotinylated probes were assessed by
standard direct ELISA methods. All nine homologous and heterologous
antigen-probe combinations were examined.
Host cell sources and cultivation. Canine MDCK epithelial cells, first
isolated from the kidney of a normal dog (17); canine DH82 histiocytes,
first isolated from a dog with malignant histiocytosis (18); and murine
C8-D1A type I astrocytes, first isolated from the cerebellums of normal
C57BL/6 mice (19), were freshly obtained from the ATCC (ATCC CCL-
34, ATCC CRL-10389, and ATCC CRL-2541, respectively). All cell lines
were cultivated as monolayers in the recommended medium supple-
mented with antibiotic-antimycotic (Cellgro, catalog no. 30-004-Cl;
Corning) in uncoated filter cap flasks and subcultured according to meth-
ods recommended by the ATCC.
Host cell colonization. MDCK, DH82, and C8-D1A cells at 50% con-
fluence on polystyrene vessel, tissue culture-treated, uncoated glass cham-
ber slides (catalog no. 354104; BD Falcon) were inoculated with live or
killed (by incubation with 10 ␮g/ml tetracycline overnight at 37°C) strains
of M. canis in 1 ml of cell culture medium; incubated under the conditions
described above; and then fixed with 4% (vol/vol) paraformaldehyde in
PBS for 10 min on ice, permeabilized with 0.1% (vol/vol) Triton X-100 for
10 min at 25°C, and blocked with 4% (vol/vol) FBS overnight at 4°C. A
sham-inoculated negative control of serum-free Dulbecco’s modified Ea-
gle’s medium (DMEM) was routinely included. The slides were probed
with primary mouse monoclonal IgG1 anti-␣-tubulin (catalog no. T9026;
Sigma-Aldrich) at a 1:500 dilution or mouse monoclonal IgG1 clone GA5
anti-glial fibrillary acidic protein (GFAP) (catalog no. 14-9892-80; eBio-
science) at a 1:500 dilution and our unbiotinylated rabbit polyclonal IgG
anti-M. canis lysate at a 1:500 dilution, coincubated for 1 h at 25°C or
overnight at 4°C. The secondary antibodies were Alexa 488-conjugated
goat anti-mouse IgG (catalog no. A11001; Invitrogen) at a 1:2,000 dilu-
tion and Alexa 546-conjugated goat anti-rabbit IgG (catalog no. A11010;
Invitrogen) at a 1:1,000 dilution, coincubated for 1 h at 25°C or overnight
at 4°C. The antibodies were diluted in an antibody diluent solution (cat-
1786 iai.asm.org Infection and Immunity
alog no. 00-3118; Invitrogen). Controls were probed with secondary an-
tibody only. Coverslips were attached by using 60 ␮l/slide of mounting
medium with 4=,6-diamidino-2-phenylindole (DAPI) (catalog no.
P36931; Invitrogen). Two-dimensional digital images were recorded by
using an EVOS FL fluorescence microscope. Z-series image stacks were
recorded at 0.2- to 0.3-␮m intervals by using a Leica TCS-SP5 confocal
fluorescence microscope and subjected to image deconvolution analyses
in three-dimensional digital reconstructions of individual infected cells by
using the Volocity Acquisition software module (Perkin-Elmer).
Preliminary time course studies were conducted to establish the rate of
cell colonization in vitro by M. canis. Adherent MDCK cells in chamber
slides were inoculated with strain PG14T at an initial multiplicity of infec-
tion (MOI) of ⬃10 mycoplasma CFU to 1 MDCK cell and then washed
and fixed for immunofluorescence imaging after 0, 2, 6, 16, 24, and 48 h of
incubation.
Stable tagging of M. canis strains PG14T, UF31, UF33, UFG1, and
UFG4 and M. cynos strain H-831T with the monomeric red fluorescent
protein mCherry was also attempted by random transposon insertion
using the mini-Tn4001-tetM plasmid vector pTF20mChloxp (see the sup-
plemental material).
Cell invasion. The capacity of M. canis strains to invade canine cells
was also assessed by using a gentamicin protection assay (20). After a cell
viability count was conducted by using trypan blue staining, MDCK and
DH82 cells were washed and resuspended in serum-free DMEM; seeded
into duplicate 6-well tissue culture-treated polystyrene plates (Costar, cat-
alog no. 3516; Corning); and then inoculated with washed M. canis strain
PG14T, UFG1, UF31, UF33, or LV cells at an initial mycoplasma CFU/
viable host cell MOI of 10:1. After 24 or 48 h of coincubation at 37°C in 5%
CO2, the medium was exchanged with serum-free DMEM plus 400 ␮g/ml
gentamicin. Controls received fresh medium without antibiotic. Incuba-
tion was continued for 3 h at 37°C in order to eradicate all extracellular
mycoplasmas. The host cells were finally washed and resuspended in se-
rum-free DMEM without antibiotic, and serial dilutions were then inoc-
ulated onto SP-4 agar to allow colonies to form from any M. canis cells that
had evaded the effects of gentamicin by intracellular invasion. The num-
bers of colonies were counted after 5 to 7 days of incubation at 37°C in 5%
CO2. Each colony was interpreted to represent one invaded host cell in
order to estimate the frequency of invasion.
Sialidase. Most strains of M. canis secrete a form of sialidase (neur-
aminidase) encoded by the nanI gene that might modulate cytadherence,
transmission, and possibly host cell injury (12). MDCK cells display ter-
minal sialic acid with both ␣-(2,3) and ␣-(2,6) linkages to subterminal
galactose in cell surface antennary oligosaccharides (21, 22), but the sur-
face glycosylation patterns of DH82 histiocytes and C8-D1A type I astro-
cytes are not well documented. The specificity of the M. canis sialidase
NanI for ␣-(2,3)- and/or ␣-(2,6)-linked sialic acid was determined by
using the lectins Maackia amurensis agglutinin (MAA), which binds to
terminal sialic acid with an ␣-(2,3) linkage to galactose, and Sambucus
nigra agglutinin (SNA), which binds to terminal sialic acid with an ␣-(2,6)
linkage to galactose, to assess desialylation of the glycoprotein substrates
fetuin (23) and transferrin (24), respectively. Triantennary fetuin includes
both ␣-(2,3) and ␣-(2,6) sialic acid linkages, while biantennary transferrin
has only the ␣-(2,6) linkage to galactose. Briefly, 5 ␮g of fetuin and trans-
ferrin was incubated separately with 2.5 ⫻ 108 CFU of washed M. canis
PG14T cells in 25 ␮l of glucose-free RPMI medium for 48 h at 37°C.
Desialylation was detected in a Western blot format by using digoxigenin
(DIG)-labeled lectin probes and alkaline phosphatase-conjugated anti-
digoxigenin polyclonal sheep Fab fragments according to the methods
provided by the lectin supplier (DIG glycan differentiation kit, catalog no.
11210238001; Roche).
Atypical M. canis strain LV lacks any detectable sialidase activity (12).
For the present studies, the genetic basis of its sialidase-negative pheno-
type was determined by whole-genome sequencing. Complete de novo
assembly of Illumina GAIIx paired-end sequencing reads from the strain
LV genome was performed by using a combination of Celera, Ray, and
June 2016 Volume 84 Number 6

Edena softwares (25–27). Annotation was accomplished via the NCBI
Prokaryotic Genome Annotation Pipeline (28). Nucleotide and amino
acid sequence similarities to the genomes of previously sequenced strains
PG14T, UF31, UF33, UFG1, and UFG4 (29) were calculated by using
JSpecies version 1.2.1 (30) and AAI Calculator (http://enve-omics.ce
.gatech.edu/aai).
The influence of host cell surface sialylation on colonization by M.
canis was assessed by using three complementary approaches. First,
MDCK cell layers in chamber slides were desialylated by preincubation for
1 h at 37°C in DMEM containing 1 U/ml of exogenous sialidase purified
from Clostridium perfringens (catalog no. N-3001; Sigma-Aldrich) (31).
The enzyme removes ␣-(2,3)-, ␣-(2,6)-, and ␣-(2,8)-linked cell surface
sialic acid, with the ␣-(2,3)-linked residues being cleaved most efficiently
(32). The cells were then washed; inoculated with strain PG14T, UF33, or
LV; and imaged in z-series stacks as described above after 24 h of incuba-
tion to minimize the potential for resialylation. Uninfected controls were
preincubated with 0, 2, 5, or 10 U/ml C. perfringens sialidase. Second, the
endogenous sialidase secreted by M. canis strain PG14T was inhibited by
preincubation of 1 ⫻ 106 CFU for 1 h at 37°C in DMEM containing 0,
0.05, 0.1, 0.5, 1, or 5 mg/ml of 2-deoxy-2,3-didehydro-N-acetyl-
neuraminic acid (DANA) (catalog no. 252926; Calbiochem), a competi-
tive inhibitor of bacterial, viral, and mammalian sialidases. The suspen-
sions of M. canis in DMEM containing DANA were then transferred onto
MDCK cell layers in chamber slides and imaged in 0.2-␮m z-series stacks
after 48 h of incubation. Uninfected controls were incubated in DMEM
containing 0 or 3.3 mg/ml DANA. Colonization rates in these two studies
were quantitated objectively by measuring the total number of anti-M.
canis antibody-labeled voxels per cell in reconstructed three-dimensional
immunofluorescence images (n ⫽ 3 random fields of view/strain, with 30
to 200 cell nuclei/field) by using a “shrink-wrapping” lasso tool to circum-
scribe precisely the total cellular volume defined by the cytoskeletal label
(Volocity Quantitation software module; Perkin-Elmer). As a third ap-
proach, untreated MDCK, DH82, and C8-D1A cells were inoculated with
sialidase-negative M. canis strain LV in chamber slides and imaged after
incubation as described above for the cell invasion studies.
Reactive oxygen species production. The potential for M. canis
strains PG14T, UF31, UF33, UFG1, and UFG4 and M. cynos strain H-831T
to produce H2O2 and possibly other reactive oxygen species (ROS) was
documented by using an assay based on the conversion of 10-acetyl-3,7-
dihydroxyphenoxazine to fluorescent resorufin in the presence of horse-
radish peroxidase in a 96-well-plate format. Briefly, 5 ⫻ 104 CFU of each
strain were pelleted by centrifugation and either resuspended immedi-
ately in a proprietary assay buffer in duplicates according to methods
provided by the supplier (catalog no. STA-344-T; Cell Biolabs) or resus-
pended in PBS and starved for 60 min at 37°C before harvest and resus-
pension in assay buffer supplemented with 100 ␮M glycerol (33). The
final reaction mixture volume was 100 ␮l. After an initial 30-min incuba-
tion at 25°C, the relative fluorescence at 590 nm was measured at 10-min
intervals for another 60 min. The amount of ROS present was estimated
by comparing the amount of resorufin accumulated in each specimen to
that generated by a series of standards containing 0 to 100 ␮M H2O2.
Cytokine expression. DH82 cells on chamber slides were inoculated
with M. canis when they had reached ~50% confluence. In trial 1, cells
were inoculated in duplicates with live cells of M. canis strain PG14T, LV,
UFG1, UF31, or UF33 that had been washed and resuspended in serum-
free DMEM without stabilized antibiotic-antimycotic at an initial myco-
plasma CFU/DH82 cell MOI of 10:1. Negative controls were sham inoc-
ulated with serum-free DMEM, and positive controls were stimulated
with 10 ␮g/ml standard lipopolysaccharide (LPS) extracted from Esche-
richia coli O111:B4 with a phenol-water mixture. Culture supernatant
liquid was harvested at 48 h postinoculation and immediately frozen in
liquid nitrogen until assayed. In trial 2, DH82 cells were inoculated in
duplicates with live or killed cells of strain PG14T or UFG1 that had been
washed and resuspended in serum-free DMEM with stabilized antibiotic-
antimycotic, at initial MOI of ~10:1 or 1,000:1 for each strain. Aliquots of
June 2016 Volume 84 Number 6 Infection and Immunity
Cellular Microbiology of Mycoplasma canis
culture supernatant liquid were harvested at 0, 24, 48, 72, and 96 h post-
inoculation and immediately frozen in liquid nitrogen until assayed. Lev-
els of gamma interferon (IFN-␥), interleukin-6 (IL-6), interleukin-10 (IL-
10), and tumor necrosis factor alpha (TNF-␣) were measured by using a
custom Milliplex canine cytokine/chemokine panel in a 96-well-plate for-
mat according to methods provided by the supplier (catalog no. CCYTO-
MAG-90K; EMD Millipore) and xMAP fluorometry systems (Luminex
LX200 in trial 1 and Bio-Plex MagPix in trial 2).
MHC-II antigen expression. DH82 cells at 50% confluence in 25-cm2
flasks were inoculated with live cells of M. canis strain PG14T, LV, UFG1,
UFG2, UFG3, or UFG4 under the conditions described above for cytokine
trial 1, including a sham-inoculated negative control of serum-free
DMEM and a positive control treated with 10 ␮g/ml LPS. After 48 h, the
cells were harvested by using a cell scraper and then washed twice with
flow wash buffer (FWB) consisting of 1 mg/ml bovine serum albumin
(BSA) plus 1 mM EDTA in PBS. Fc-binding receptors specific for IgG2a
(34) were blocked in FWB plus 10% (vol/vol) FBS or rat serum (catalog
no. R-9759; Sigma-Aldrich) for 15 min at 4°C, and duplicates of ⬃1 ⫻ 106
cells were then resuspended in 90 ␮l of FWB plus 10 ␮l (0.5 ␮g) of Alexa
647-conjugated rat IgG2a monoclonal anti-canine major histocompati-
bility complex class II (MHC-II) antibody (clone YKIX334.2) (catalog no.
51-5909; eBioscience) (35) and incubated in the dark for 1 h at 25°C.
Isotype controls were incubated with the same concentration of an Alexa
647-conjugated mouse (catalog no. 51-4724; eBioscience) or rat (catalog
no. 557690; BD Pharmingen) IgG2a⌲ monoclonal antibody against key-
hole limpet hemocyanin (KLH). After three washes in FWB, the cells were
resuspended in 4% (vol/vol) paraformaldehyde for 10 min at 4°C and
then washed, resuspended in 250 ␮l of FWB, and strained through
prewetted 35-␮m nylon mesh into round-bottom polystyrene tubes (cat-
alog no. REF352235; Falcon) for flow cytometry (36). The fluorescence
intensity of 5 ⫻ 104 to 1 ⫻ 105 events (cells) was measured for each
specimen.
Endothelin-1 and complement factor H expression. DH82 cells at
50% confluence in 25-cm2 flasks were inoculated with live cells of M. canis
strain PG14T, UF31, UF33, LV, or UFG1 under the conditions described
above for MHC-II expression, including a sham-inoculated negative con-
trol of serum-free DMEM and a positive control of 10 ␮g/ml standard
LPS. The culture supernatant was harvested at 48 h postinoculation and
immediately frozen in liquid nitrogen until assayed. The levels of the
vasoconstrictive and neuroendocrine regulatory peptide endothelin-1
(ET-1) (37, 38) and canine complement factor H were quantitated by
using colorimetric sandwich ELISAs according to methods recommended
by the suppliers (R&D Systems [catalog no. DET100] and NovaTeinBio
[catalog no. BG-CAN10534], respectively).
Statistical analyses. The effects of the above-described treatments on
colonization rates and expression levels of cytokines, ET-1, and comple-
ment factor H were analyzed by analysis of variance (ANOVA) using JMP
Pro v11.0 (SAS Institute) and by post hoc Tukey-Kramer honestly signif-
icant difference (HSD) or Dunnett comparisons to controls when main
effects were significant (P ⬍ 0.05). The relative levels of MHC-II expres-
sion by infected DH82 cells were compared to those of uninoculated con-
trols by using Kolmogorov-Smirnov and probability binning statistical
algorithms (FlowJo v9.6; BD Biosciences). A ␹(T) value of ⬎4 was consid-
ered significant (39).
Immunohistology. Formalin-fixed, paraffin-embedded brain tissue
sections from three archived cases of NME and three archived cases of
GME were processed for immunohistochemical staining by using the
biotinylated polyclonal anti-M. canis strain PG14T probe described above.
Controls were fixed brain tissue sections from archived canine cases of
idiopathic cardiac necrosis, chronic hepatopathy, and hemangiosarcoma.
The slides were deparaffinized and rehydrated by standard methods, in-
cluding an intermediary step to quench endogenous peroxidase activity
(3% H2O2 in methanol). For enzyme-induced antigen retrieval, sections
were submerged in a proprietary trypsin solution (catalog no. 00-3006;
Invitrogen). For heat-induced antigen retrieval, sections were heated in a
iai.asm.org 1787
Michaels et al.
steamer while being submerged in a neutral-pH EDTA solution (catalog
no. 920P; Cell Marque). Slides were then coincubated with a universal
blocking reagent (catalog no. BS966; Biocare Medical) and a polyclonal
anti-M. canis probe at a 1:2,000, 1:4,000, or 1:8,000 dilution for 16 h at
40°C, followed by application of a peroxidase-conjugated secondary an-
tibody (catalog no. RHRP520; Biocare Medical). Bound probe was de-
tected by incubating slides in 3=,3=-diaminobenzidine (DAB) (catalog no.
DB801; Biocare Medical). Slides were counterstained with hematoxylin,
and coverslips were mounted by using an antioxidant medium (catalog
no. 8312-4; Thermo Scientific). The entire surface of each slide was in-
spected for accumulation of oxidized DAB precipitates.
RESULTS
Genomics and transformation. The closed circular M. canis
strain LV genome assembly (GenBank accession no. CP011368.1)
was 968,791 bp in length and had 27% G⫹C content, with 97 to
98% average nucleotide identity and 98.1 to 99.5% predicted
amino acid identity to five other strains of M. canis. The tet-
ranucleotide frequency correlation with the other strains was
0.9955 to 0.9957. This is the first complete genome of M. canis. A
1.9-kb insertion evident in the sialidase homolog nanI was con-
firmed by PCR-based Sanger sequencing. It consisted of an inser-
tion sequence (IS) encoding a 509-amino-acid (aa) isoform of a
streptococcal IS1202 group transposase (blastp E value of 2e⫺16)
flanked by perfect 90-nucleotide (nt) direct repeats, one of which
also occurs in the wild-type nanI open reading frame (ORF) of
PG14T and other strains of M. canis. This insertion introduced
multiple premature stop codons into the nanI ORF of strain LV,
which was sufficient to explain its sialidase-negative phenotype.
The transposase had regions of very high similarity (blastp E val-
ues of 1e⫺48 to 4e⫺91) to two series of short predicted ORFs,
MYCN0660 to MYCN0662 and MYCN0767 MYCN0769, anno-
tated in the genome of the canine pathogen M. cynos strain C142
(GenBank accession no. HF559394.1). M. cynos strain H-831T was
also PCR positive for the transposase, whereas 10 other isolates of
M. canis and M. arginini strain G230T, M. bovigenitalium strain
PG11T, M. edwardii strain PG24T, M. maculosum strain Skotti B,
M. molare strain H542T, M. opalescens strain MH5408T, and M.
spumans strain PG13T were PCR negative, providing evidence that
the sialidase-negative phenotype of M. canis strain LV results from
insertional inactivation by an IS acquired naturally from M. cynos,
possibly during coinfection of a canine host. No other relevant
differences from the genomes of other strains of M. canis were
recognized.
All strains of M. canis, and M. cynos H-831T, resisted stable
genetic transformation with IS256-based random transposition
vectors under a multitude of polyethylene glycol (PEG), Lipofec-
tin, Lipofectamine, and electroporation conditions (see the sup-
plemental material). However, the presence of an IS1202 group
transposase isoform in the genome of strain LV suggests that M.
canis may be amenable to transformation with IS1202-based con-
structs in the future.
Colonization and invasion. Under standard SEM conditions,
cells of strain PG14T were coccoid, with a diameter of ⬃350 nm,
which is ⬃1/25 of the diameter of a typical MDCK cell nucleus.
They had a lightly textured surface marked by 20-nm circular
elevations occurring in a pattern of stripes along the axis of the
cells (see Fig. S1 in the supplemental material). No quantitative
differences in binding to M. canis whole-cell lysate antigens of
different strains, or qualitative differences in the labeling pattern
of cells of different M. canis strains adherent to host cells in vitro,
1788 iai.asm.org Infection and Immunity
A B
C D
FIG 1 Cell colonization and invasion by M. canis PG14T. (A and B) Crosshairs
indicate perinuclear (A) and intracytoplasmic (B) immunolabeled M. canis
cells (red) in MDCK epithelial cells optically sectioned in the z-axis at 0.2-␮m
intervals. The cytoskeleton was labeled with anti-␣-tubulin (green), and nuclei
were labeled with DAPI (blue). (C and D) Similar patterns of colonization
were observed with DH82 histiocytes labeled with anti-␣-tubulin (C) and with
C8-D1A astrocytes labeled with anti-glial fibrillary acidic protein (D).
were observed among the polyclonal antibody reagents generated
for these studies, so the anti-PG14T antibody was adopted as the
standard probe. In preliminary time course studies, little surface
colonization of MDCK or DH82 cells was apparent microscopi-
cally before 24 h postinoculation, and the background of non-
cytadherent M. canis cells increased substantially beyond 48 h
postinoculation, so 48 h was adopted as the standard incubation
period for most subsequent experiments. For all strains examined,
M. canis was most often observed attached at the margins of host
cells in monolayers or in close proximity to host cell nuclei (Fig.
1A to D). Intracytoplasmic M. canis cells (Fig. 1B) were also evi-
dent in three-dimensional image reconstructions of infected host
cells of all types. Complementary evidence of host cell invasion
was the recovery of viable cells of strains PG14T, UF31, and UF33
as early as 24 h postinoculation from both MDCK and DH82 cells
subsequently treated with gentamicin (Table 1). Cells of strains
PG14T, UF31, and UF33 were recovered from the interior of about
1 of every 10 host cells inoculated, while cells of strains LV and
UFG1 were not recovered from either MDCK or DH82 cells fol-
lowing gentamicin treatment. The remains of killed M. canis cells
also bound extensively to host cells (see Fig. S2 in the supplemen-
tal material).
Role of sialidase in colonization. Sialidase-negative strain LV
colonized untreated MDCK cells as efficiently as wild-type strain
PG14T did, and quantitative strain differences (P ⬍ 0.05) in the
extents of colonization either with or without prior desialylation
were at most 2-fold (Fig. 2A). However, pretreatment with exog-
enous C. perfringens sialidase to desialylate the surfaces of MDCK
cells significantly reduced (P ⬍ 0.01) their colonization by all
June 2016 Volume 84 Number 6

TABLE 1 Intracellular invasion and persistence of Mycoplasma canis in cultured canine cellsa
Mean CFU in canine cells
⫺ gentamicin
24 h 48 h
Strain MDCK DH82 MDCK
PG14T 1.1 ⫻ 103 7.0 ⫻ 102 1.5 ⫻ 103
UFG1 1.5 ⫻ 104 1.5 ⫻ 104 1.6 ⫻ 104
LV 8.8 ⫻ 102 8.5 ⫻ 102 9.4 ⫻ 103
UF31 9.9 ⫻ 103 1.6 ⫻ 104 1.1 ⫻ 104
UF33 2.1 ⫻ 104 2.0 ⫻ 104 2.2 ⫻ 104
Pooled SEM 3.9 ⫻ 103 4.1 ⫻ 103 3.4 ⫻ 103
a
Values are mean CFU (n ⫽ 2) recovered from 1 ⫻ 10 viable nonphagocytic MDCK epithelial or phagocytic DH82 histiocyte cells in serum-free medium inoculated with washed
5
M. canis cells at an MOI of 10. One CFU represents one infected host cell. Times shown are hours of coincubation of M. canis with canine cells before exposure to 400 ␮g/ml
gentamicin for 3 h at 37°C to eradicate extracellular mycoplasmas.
strains tested 24 h after inoculation (Fig. 2A). Furthermore, coin-
cubation of strain PG14T with DANA, a competitive inhibitor of
its endogenous sialidase, significantly enhanced (P ⬍ 0.05) the
number of M. canis cells remaining attached to untreated MDCK
cells 48 h after inoculation in a concentration-dependent fashion
(Fig. 2B). The size, shape, and number of adherent uninfected
control cells were unaffected by the above-described treatments
with C. perfringens sialidase or DANA.
The secreted sialidase of M. canis was shown to have a prefer-
ence for terminal sialic acid with an ␣-(2,3) linkage to subterminal
galactose when binding of MAA lectin to fetuin was completely
abolished by incubation of the ligand with M. canis cells (Fig. 2C).
Less transferrin was modified by incubation with M. canis, as ev-
idenced by the persistence of SNA binding to a predominant 65-
kDa band on Western blots and by the appearance of a new 45-
kDa band indicating the removal of only one of transferrin’s two
terminal sialic acid residues with an ␣-(2,6) linkage to subterminal
galactose. M. canis NanI therefore had the same linkage specificity
and relative cleavage efficiency as those of the sialidase purified
from C. perfringens.
Reactive oxygen species. The rate of ROS excretion was strain
variable, with the majority of nonstarved cells of the M. canis
strains generating a minor increase in excretion in the assay me-
dium, equivalent to ⬍2 ␮M H2O2/105 CFU/h, although a few
strains, including brain isolate G1, generated 3- to 5-fold more
ROS (P ⬍ 0.05) (Fig. 3). Endogenous glycerol-3-phosphate was
the presumed substrate for glycerol-3-phosphate oxidase-depen-
dent H2O2 production under these conditions, because following
starvation before the assay, no strain of M. canis excreted detect-
able ROS into glycerol-supplemented assay medium (data not
shown).
Cellular responses to colonization. When measured at 48 h
postinoculation, strains of M. canis varied significantly (P ⬍ 0.01)
in the extent to which they influenced the in vitro secretion of
TNF-␣ by DH82 histiocytes, while the effect of strains did not
reach statistical significance for the secretion of IFN-␥, IL-6, or
IL-10 (Fig. 4A to D). Vaginal isolates UF31 and UF33 evoked a
Th1-type proinflammatory TNF-␣, IL-6, and IFN-␥ profile,
whereas PG14T, LV, and especially brain isolate UFG1, in contrast,
seemed anti-inflammatory, with high IL-10/IFN-␥ ratios and the
smallest amount of TNF-␣ elicited. The concentration of IFN-␥ in
the cell culture medium did not increase (P ⬎ 0.05) beyond 48 h of
June 2016 Volume 84 Number 6
Cellular Microbiology of Mycoplasma canis
⫹ gentamicin
24 h 48 h
DH82 MDCK DH82 MDCK DH82
8.8 ⫻ 102 0 0 6.3 ⫻ 102 8.5 ⫻ 102
1.5 ⫻ 104 0 0 0 0
3.3 ⫻ 103 0 0 0 0
2.0 ⫻ 104 4.1 ⫻ 103 8.6 ⫻ 102 4.3 ⫻ 103 4.9 ⫻ 103
2.4 ⫻ 104 3.6 ⫻ 103 1.1 ⫻ 103 3.8 ⫻ 103 3.3 ⫻ 103
4.6 ⫻ 103 3.0 ⫻ 102 1.0 ⫻ 102 1.1 ⫻ 103 1.2 ⫻ 103
incubation with strain PG14T or UFG1, but TNF-␣, IL-6, and
IL-10 concentrations continued to increase steadily (P ⬍ 0.01)
through 96 h. A similar time course of responses for TNF-␣, IL-6,
and IL-10 was observed when the MOI was increased to 1,000:1,
although the concentrations of all cytokines measured were unex-
pectedly lower (P ⬍ 0.01) at the higher MOI. Exposure of DH82 cells
to killed cells of strain PG14T or UFG1 had a tendency to induce
slightly more (P ⬍ 0.10) IL-10 at each time point than inoculation
with live M. canis cells did, and killed cells elicited the secretion of
as much of the other cytokines as live M. canis cells did.
Histiocyte MHC-II antigen expression was uniformly sup-
pressed (P ⬍ 0.01) by all strains, with 40 to 60% decreases in the
median fluorescence intensity induced by strains PG14T and LV
(Fig. 5) and 15 to 45% decreases induced by the other strains
tested. The median fluorescence intensity of uninoculated con-
trols incubated in 10 ␮g/ml LPS increased 12% (P ⬍ 0.01). No
effects of the type of blocking serum (bovine versus rat) or amount
of primary antibody (0.05 to 1.25 ␮g/106 DH82 cells) were de-
tected, but uninoculated histiocytes exposed to the mouse isotype
control monoclonal antibody had consistently greater nonspecific
fluorescence than did those exposed to the rat isotype equivalent.
Secretion of ET-1 by DH82 cells was inversely related to TNF-␣
and IFN-␥ production, with the greatest decrease in ET-1 (P ⬍
0.05) being elicited by strain UFG1 and only slight decreases being
elicited by UF31 and UF33 (Fig. 6). The secretion of complement
factor H (3.43 ⫾ 0.05 ng/ml of medium after 48 h for 8 untreated
controls) was not significantly affected by inoculation of DH82
cells with any strain of M. canis or by incubation in 10 ␮g/ml LPS
(range, 0.5 to 4.0 ng/ml of medium).
No in vitro cytopathic effects of colonization (rounding, bleb-
bing, vacuolization, or pycnosis) were observed under any condi-
tions for MDCK, DH82, or C8-D1A cells inoculated with any
strain of M. canis (Fig. 1).
Immunohistology. Preliminary studies of NME lesion posi-
tive-control serial sections showed that neither trypsin nor heated
EDTA antigen retrieval treatment affected the DAB staining pat-
terns. The final method adopted employed no antigen retrieval
and a probe dilution of 1:8,000 (1 ␮g/ml of biotinylated polyclonal
anti-M. canis strain PG14T probe). Oxidized DAB precipitates
were never seen in negative-control brain specimens (Fig. 7A), but
a focal diffuse precipitate was occasionally evident in case speci-
mens (Fig. 7B and C).
Infection and Immunity iai.asm.org 1789
Michaels et al.

140 15
A Untreated Sialidase-treated D
B
120

H2O2 (Δ μM/105 CFU/hr)

C
10

100 B
Voxels / MDCK cell

80 5

A,B A A
60
0

yn
4

G2
G3
G4
31
33

26

G1
LV

5
M l
Ca
ar
1

.c
PG
40

M
D
C,D Strain
20 C FIG 3 Strain-variable excretion of ROS by freshly harvested M. canis cells in
glycerol-free assay medium. The rate of excretion of H2O2 and possibly other
ROS was measured by using an assay based on the oxidation of 10-acetyl-3,7-
0 dihydroxyphenoxazine to fluorescent resorufin. Means (⫾ standard errors of
PG14 LV 33 PG14 LV 33 the means) with different superscripts differ (P ⬍ 0.05 by Tukey-Kramer HSD
Strain post hoc comparisons). Following starvation for 1 h before the assay, no strain
excreted detectable ROS in glycerol-supplemented assay medium (data not
1200 shown). M. cyn, Mycoplasma cynos strain H-831T.
B C
250
B 150
1000
100
meningitis pathogens Haemophilus, Streptococcus, and Neisseria
Controls

75
(41, 42). Fatality rates, however, are highest among the cases ulti-
800 mately attributed to atypical agents, including species of Myco-
Voxels / MDCK cell

50

37
plasma and other slow-growing fastidious organisms that resist
600 standard empirical treatment with cell wall-targeting antibiotics
250 (10, 43–46). Bacterial neuropathogens vary in their mechanisms
150
A 100 of virulence, but assumptions regarding the importance of phago-
M. canis

400 A 75
cytosis/exocytosis, fimbriae, capsules, peptidoglycan, and LPS for
50 epithelial penetration and development of bacteremia associated
200 with ME (47–50) are all invalid regarding the pathogenesis of my-
37
coplasmal diseases (51, 52). This is important because the diag-
nostic uncertainty and empirical treatment failures that precede
A rrin

+ tuin
r
ke

AA

0
SN fe
ar

the adverse outcomes of many atypical infections cannot be ex-

fe
M
+ ns
m

0 5 50
tra

DANA (mg/ml) pected to improve without more comprehensive knowledge of the

FIG 2 Role of sialidase in M. canis colonization. (A) Pretreatment of MDCK
cells with exogenous sialidase reduces the extent of colonization by all strains
tested. Endogenous sialidase-negative strain LV colonized as efficiently as
wild-type strain PG14T did. (B) Coincubation with DANA, a competitive in-
hibitor of sialidase, enhances the number of M. canis cells remaining attached
to MDCK cells 48 h after inoculation in a concentration-dependent fashion.
Means (⫾ standard errors of the means) with different superscripts differ (P ⬍
0.05 by Tukey-Kramer HSD post hoc comparisons). (C) Binding of MAA lectin
to fetuin is completely abolished by incubation of the ligand with M. canis,
showing that the secreted sialidase of M. canis efficiently removes terminal
sialic acid linked with ␣-(2,3) to galactose. Comparatively less transferrin was
modified by incubation with M. canis, as evidenced by the persistence of SNA
lectin binding to a predominant 65-kDa band on Western blots and by the
appearance of a new 45-kDa band indicating the removal of only one of trans-
ferrin’s two ␣-(2,6)-linked terminal sialic acid residues. The blots were spliced
for labeling purposes.
DISCUSSION
The adult human mortality rate of 20 to 30% due to bacterial
meningoencephalitis has not been significantly reduced by mod-
ern intensive care, diagnostics, or therapy (40). Sequelae among
survivors include paresis, epileptic seizures, cerebral palsy, deaf-
ness, and cognitive deficits. Gaps in understanding bacterial ME
reflect the prevailing research and clinical emphasis on the classic
etiologic agents involved (6, 44, 53, 54).
Several strains of M. canis readily colonized the surfaces of cells
integral to epithelial and innate immune barriers to dissemina-
tion, as well as one type of brain cell (Fig. 1). M. canis adheres to
sialylated receptors on host cells (55). The extent of initial coloni-
zation was reduced by pretreatment to remove ␣-(2,3)- and
␣-(2,6)-linked sialic acid from host cell surfaces, but the sialidase
secreted by M. canis itself did not limit cytadherence to untreated
cells, as shown by the similarity in adherences of wild-type strain
PG14T and sialidase-negative strain LV (Fig. 2A). The enhanced
amount of cells of PG14T remaining attached to untreated MDCK
cells after incubation in the presence of the sialidase inhibitor
DANA (Fig. 2B) is evidence that its sialidase may instead be im-
portant primarily for detachment from colonized host cells and
subsequent transmission. The enzyme’s preference for ␣-(2,3)-
linked substrates (Fig. 2C) supports the conclusion that the en-
zyme adheres predominantly to ␣-(2,6)-linked sialoreceptors.
The proportional expression of this glycosylation pattern at dif-
ferent anatomical sites, as well as strain differences in the amounts
of sialidase secreted (12), could influence host species range, pri-
mary colonization of respiratory or urogenital tract mucosal sur-
faces, and dissemination and secondary localization of M. canis
cells in an infected host.

1790 iai.asm.org Infection and Immunity June 2016 Volume 84 Number 6

 Cellular Microbiology of Mycoplasma canis

A B 300
2000
C
250

1500

TNFα (pg/ml)
200

IL-10 (pg/ml)
B
A
150
1000

100
500
50

0 0
M PG14 LV G1 31 33 LPS M PG14 LV G1 31 33 LPS
Strain Strain

C 1200 D 70
1000 60

50
800

IFNγ (pg/ml)
IL-6 (pg/ml)

40
600
30
400
20

200 10

0 0
M PG14 LV G1 31 33 LPS M PG14 LV G1 31 33 LPS
Strain Strain
FIG 4 Cytokine responses of DH82 histiocytes to colonization with M. canis. Data shown are from 48 h after inoculation with live M. canis cells. M, untreated
controls; LPS, controls incubated with 10 ␮g/ml E. coli LPS. Means (⫾ standard errors of the means) with different superscripts differ (P ⬍ 0.05 by Tukey-Kramer
HSD post hoc comparisons).

The presence of visible M. canis inside nonphagocytic cells and
its recovery following eradication of extracellular bacteria from
both nonphagocytic and phagocytic cells demonstrated its innate
capacity for intracellular invasion and persistence (Fig. 1A and B
and Table 1). This finding established the potential for direct pen-
etration through adjacent tissues, versus strictly “Trojan horse”
spread via infected macrophages (56, 57), as a basis for the dissem-
ination of M. canis from mucosal surfaces. However, the absence
of cytopathic effects on any type of host cell colonized in vitro was
evidence against direct injury by M. canis and was in favor of
multifactorial host responses to colonization as the potential basis
for any role in ME or other diseases.
In canine NME, pathological findings consist of neuroparen-
chymal infiltration of lymphocytes and monocytes or histiocytes,
with malacia and necrosis usually being localized in the cerebral
cortex and subcortical region (58–61). Similar pathologies have
been attributed to other species of mycoplasmas that gained entry
into the brain (62–64). The profile of cytokine secretion by DH82
cells following exposure to M. canis isolates UF31 and UF33,
characterized by high TNF-␣/IL-10 ratios, clearly contrasted

100
Isotype Uninfe
f cted A
50
B
Endothelin-1 (pg/ml)

80 C
% of maximum

40 D
60 LV E
PG14 30

40
20

20
10

0 0
10 2 10 3 10 4 10 5 M 31 33 PG14 LV G1 LPS
MHC-II fluorescence Strain
FIG 5 Effects of M. canis on MHC-II antigen expression by DH82 histiocytes. FIG 6 Strain-variable effect of M. canis on secretion of endothelin-1 by DH82
Decreases in the median fluorescence intensity of 40 to 60% were induced by histiocytes. M, untreated controls; LPS, controls incubated with 10 ␮g/ml E.
strains PG14T and LV, while 15 to 45% decreases were induced by the other coli LPS. Means (⫾ standard errors of the means) with different superscripts
strains tested [P ⬍ 0.01 by ␹(T) test]. differ (P ⬍ 0.05 by Tukey-Kramer HSD post hoc comparisons).

June 2016 Volume 84 Number 6 Infection and Immunity iai.asm.org 1791

Michaels et al.
FIG 7 Immunohistochemical staining of archived NME and GME brain tissue samples probed with polyclonal anti-M. canis strain PG14T antibody. Bound
probe was detected with a peroxidase-conjugated secondary antibody and the DAB substrate. Slides were counterstained with hematoxylin (violet). (A)
Negative-control canine brain parenchyma (magnification, ⫻10); (B) focal deposition of oxidized DAB precipitate (brown) throughout a vascular cuff in NME
tissue (magnification, ⫻10); (C) detail from panel B (magnification, ⫻40).
with the responses to strains PG14T, LV, and UFG1, character-
ized by high IL-10/IFN-␥ ratios (Fig. 4A to D). This showed
that M. canis is capable of eliciting either pro- or anti-inflam-
matory host responses in a strain-dependent fashion, which
might influence the network of immune barriers to dissemina-
tion and recruitment and modulation of inflammatory cells at
secondary sites (65). Interestingly, exposure to killed M. canis
cells elicited cytokine secretion as effectively as exposure to live
M. canis cells did, suggesting that diacylated membrane lipopep-
tides are the activating factors (66–71). The principal pathogenic
effect of exposure to individual mycoplasmal lipoproteins charac-
terized to date is transient inflammation (72), marked by local
infiltration of granulocytes, macrophages, and lymphocytes; the
production of proinflammatory cytokines; and complement acti-
vation (73–75).
The consistently reduced expression of MHC-II observed (Fig.
5) is evidence that the presentation of M. canis or other antigens to
CD4⫹ lymphocytes could be locally compromised during myco-
plasmal dissemination or by M. canis at secondary sites of infec-
tion. This effect on cellular immunity would further contribute to
general immune dysregulation by strains, such as brain isolate
UFG1, that elicit comparatively low IFN-␥ or TNF-␣ responses.
This pattern is consistent with DH82 cell MHC-II receptor down-
regulation caused by infection with the obligate intracellular ca-
nine pathogen Ehrlichia canis (36) and with the paucity of MHC-
II-expressing macrophages in necrotic joint lesions of calves
infected with Mycoplasma bovis by intra-articular challenge (76)
but contrasts with the marked upregulation of MHC-II-express-
ing airway epithelial cells following intranasal inoculation of rats
with Mycoplasma pulmonis (77).
ET-1 is a potent vasoconstrictor and also regulates central au-
tonomic control of circulation, respiration, sympathetic vasomo-
tor discharges, and the hypothalamic-pituitary axis (78). M. canis
significantly reduced DH82 cell expression of ET-1 (Fig. 6), in
contrast to the increases typically resulting from exposure to other
bacteria (37, 79–81). Once more, the effect was greatest for brain
isolate UFG1. This finding showed yet again how M. canis might
influence ME in ways that differ from those of classic neuropatho-
gens (82). For example, increased ET-1 levels during acute pneu-
mococcal sepsis promote meningitis through vasoconstriction,
resulting in cerebral ischemia (81, 83). Brain endothelial cells,
microglia, astrocytes, and neurons are other sources of ET-1 (78,
84). We observed endogenous secretion levels comparable to
those found in DH82 cells by Divino et al. (37), but we did not
1792 iai.asm.org Infection and Immunity
observe the reported stimulation with LPS. Methodological dif-
ferences from that study included DH82 passage levels (85), the
preparation of LPS (E. coli O111:B4 versus O127:B8), and end-
point after stimulation (48 h versus 24 h or less).
Canine GME is distinguished from NME by perivascular
cuffing with lymphocytes, macrophages, and neutrophils as
well as granulomatous lesions mainly in the cerebellum and
brainstem, but both NME and GME are believed to share a
pathogenesis (61). Although it did not occur frequently or con-
sistently among cases of GME or NME, deposition of oxidized
DAB throughout some vascular cuffs probed with anti-M. canis
antibody (Fig. 7B and C) was evidence that the presence of M.
canis in canine brains cannot be attributed solely to contami-
nation during nonaseptic tissue collection (2). Because brain
tissue specimens are typically not available for examination
until long after clinical signs of chronic progressive neurolog-
ical disease have developed, the abundance of intact M. canis
cells remaining to be visualized at necropsy may be very low. A
different polyclonal anti-M. canis antibody did not detect intact
M. canis cells in similar cases examined (2).
In conclusion, no acute cytopathic effects of in vitro coloniza-
tion of any host cell type or consistent patterns of M. canis poly-
valent antigen distribution in canine ME case brain tissues were
evident in the present studies. A causal role of M. canis in canine
ME thus remains to be demonstrated. This work was limited by
the paucity of commercially available canine-specific cell lines and
analytical reagents and by the range of MOIs, analytes, and end-
points that were practical to sample in the in vitro infection stud-
ies. The specimens for immunohistology were not matched with
unfixed tissues that could be tested by culture or PCR, and they
represented only a tiny fraction of each brain. Any influence of M.
canis on ME probably involves interactions within the complex
multicellular and neurochemical milieu in vivo (86), which could
be explored in an organotypic tissue explant model (87–89), but a
prospective survey for M. canis in aseptically sampled cases of ME
is needed. Positive impacts can be expected to include not only a
better understanding of the ordinarily commensal organism M.
canis and its role in canine diseases but also further insights into
processes of bacterial invasion of the central nervous system
(CNS) and subsequent injury, which is necessary to develop more
comprehensive diagnostics and therapeutic interventions to re-
duce the burden of human and veterinary infectious neurological
disease.
June 2016 Volume 84 Number 6

ACKNOWLEDGMENTS
Strains UF31, UF33, LV, 5, 26, Cal, and Mara were provided by Mary
Brown at the University of Florida (UF). DNA sequencing and antibody
purification and labeling were performed by the UF Interdisciplinary
Center for Biotechnology Research. Electron microscopic imaging was
performed at the Miami University Center for Advanced Microscopy and
Imaging. Brian Porter (Texas A&M University, College Station, TX, USA)
provided clinical case and control specimens for immunohistology. We
gratefully acknowledge technical assistance at UF from Anthony Barbet
(bioinformatics), Ann Fu (immunohistology), Craig Moneypenny (im-
munofluorescence microscopy and flow cytometry), and Marc Salute (cy-
tokine assays). We thank Scott Schatzberg (Veterinary Emergency & Spe-
cialty Center, Santa Fe, NM, USA) for advice and critique throughout this
study.
We declare no conflicting interests.
The funders had no role in study design, data collection and interpre-
tation, or the decision to submit the work for publication.
FUNDING INFORMATION
This work, including the efforts of Daniel R. Brown, was funded by Harold
and Vera Morris Trust Research Fund. This work, including the efforts of
Daniel R. Brown, was funded by University of Florida College of Veteri-
nary Medicine Faculty Research Development Fund. This work, including
the efforts of Jeffrey A. Leibowitz, was funded by University of Florida
University Scholars Program.
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