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Mycoplasma Hyorhinis Swine Arthritis. I. Clinical and Microbiologic Features

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arthritis
and
rheumatism
~~
Official Journal of The American Rheumatism Association
Section of The Arthritis Foundation
Mycoplasma Hyohinis Swine Arthritis
1. Clinical and Microbiologic Features
Jerri A. Barden and John
L. Decker
An acute arthritis is followed in 6 to 8 months by the development of chronic
polyarthritis when young Yorkshire swine are given a single intraperitoneal
inoculation of Mycoplasma hyorhinis. Sequential antibody studies, using the
metabolic inhibition test, revealed a brisk response in infected animals followed
by sustained elevations of titers. Synovial fluid titers were found to exceed those
of serum, suggesting local antibody production. The organism, readily recovered
in the acute phase, became progressively more difficult to isolate.
The weight of evidence today suggests
that rheumatoid arthritis (RA) is an infection (1,2). Model diseases of known microbial etiology and with articular lesions
comparable to those of RA are thus of
interest. Experimental Mycoplasma hyorhinis arthritis of swine appears to conform to this description (3).
The process, to which only young aniFrom the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases,
and Arthritis and Rheumatism Branch, National
Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md 20014.
JERRI A. BARDEN, MD:
Medical Officer, Health
Services and Mental Health Administration, Rockville, Md. JOHN L. DECKER, MD: Chief, Arthritis
and Rheumatism Branch, National Institute of
Arthritis and Metabolic Diseases, Room 9N218,
Building 10, NIH, Bethesda, Md.
Requests for reprints should be addressed to
Dr. Decker.
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
mals are said to be susceptible, results in a
polyarthritis characterized by mononuclear
cell infiltration of the synovia. The experimental studies available indicate the organism persists in the joints for up to two
months after peritoneal inoculation (4),
but natural cases with arthritis of longer
duration have been reported to have sterile
synovial fluid (5). No information on the
animals’ sequential antibody response to
the infecting organism is available, although the metabolic inhibition technique
of Taylor-Robinson et a1 (6) appears capable of providing such.
It seemed to us important to assess the
clinical process, the recoverability of the
organism, and the presence of antibodies in
a model disease where. in contrast to RA,
the microbial stimulus or antigen is known.
This report describes a pilot study of the
193
BARDEN 81 DECKER
experimental disease through 8 months.
The pathologic findings are detailed in an
accompanying report (7).
MATERIALS AND METHODS
Swine
Twenty-two Yorkshire swine were delivered by
hysterectomy and raised in a specific pathogen-free
(SPF) laboratory. They were removed from their
SPF environment after 3 to 4 weeks and paired by
sex. weight and litter into two groups, which were
strictly isolated from one another. Both groups
were housed on concrete floors in similar environments with constant temperature, and fed antibiotic-free feed ad libitum.
0bservations
Clinical observations were made at least 3 times a
week on ail animals beginning one week prior to
infection. These included general appearance, behavior and appetite as well as findings pertinent to
the musculoskeletal system with particular emphasis on gait and individual joint involvement.
Rectal temperatures were recorded. Body weights
were determined weekly.
Clinical Laboratory Studies
Blood was taken weekly for hematologic and
biochemical studies. Hematocrit (HCT),white
blood cell (WBC). and differential count, erythrocyte sedimentation rate (ESR) by the Westergren
method, total serum protein ( 8 ) . serum glutamic
oxaloacetic transaminase (SGOT) (9) , alkaline
phosphatase (10). blood urea nitrogen (BUN),
total serum bilirubin (11) and Serum glucose (12)
were performed. Serum electrophoresis was run,
stained and analyzed by Beckman analyotron. T h e
bentonite flocculation test @FT) was performed
using particles sensitized with either human or
porcine 7 globulins (13). Cold agglutinins were
sought (14).
Microbiologic Studies
Nose swabs and blood cultures were taken prior
to infection and on a weekly basis thereafter for the
culture of M hyorhinis. T h e cotton-tipped swabs
were streaked directly onto agar and then twirled in
broth. Agar and broth both contained 70% beef
heart infusion broth,. 20% horse serum (BBL),?
+Difco Laboratories, Detroit, Mich.
tMicrobiologica1 Associates Inc, Bethesda, Md.
194
10% fresh yeast extract,$ and bacterial inhibitors
(15). T h e broth medium was supplemented with
1% glucose and 0.002% phenol red. Blood cultures
were taken from the superior vena cava and 1 ml of
blood was inoculated into 20 ml of broth.
At the time of sacrifice, mycoplasma cultures
were taken aseptically from the abdomen, thorax,
central nervous system (CNS) and joints. In all
cases culures were taken directly on agar and,
where possible, both broth and agar were employed. Tissue aliquots varying from 0.5 to 3.0 cm
in diameter were minced and inoculated into 20 ml
of broth. Where fluids were obtained, 0.05-0.1 ml
was used as the inoculum. Blood and tissue broth
tubes were incubated at 370 C for 4 days and then
plated onto agar. Titrations were made where
possible. All plates were read between 4 and 14
days. Any mycoplasma colonies found were stained
with fluorescein-conjugated antisera to M hyorhinis
obtained from the Division of Biological Standards
and used a t a 1:40 dilution (16).
Weekly sera were examined for growth inhibiting
activity against M hyorhinis strain 7, as measured in
the metabolic inhibition test (MIT) (6). Where
possible, synovial fluids were also analyzed for the
presence of antibody. M pneumoniae and M granulurum were used as controls in the MIT.
Preparation of lnoculum and
Infection of Herd
M hyorhinls. Strain 7, pass 40 (obtained from
Dr. William Switzer. Ames, Iowa), was passed twice
i n beef heart infusion media with 20% turkey
serum, 0.2% bacteriologic hemoglobin and 0.5%
swine gastric mucin, to obtain a pool suitable for
egg passage. One-tenth milliliter of this pool. containing approximately 10s colony-forming units per
milliliter (CFU/ml) , was used to inoculate each 6to 8-day-old chick embryo. Fluid was collected from
dead embryos 6 to 10 days postinfection. Harvested
material from each chick was tested on blood agar
plates and in thioglycollate media for bacterial
contamination and subsequently pooled. The pool
was again tested on blood agar (aerobically and
anaerobically) , thioglycollate, Sabouraud chocolate
agar and on cystine dextrose blood chocolate agar.
I t contained approximately l W CFU/ml M hyorhinis. T h e organism was typed with the MIT, using
hyperimmunized rabbit anti-M hyorhinis sera. Agar
colonies were stained with fluorescein-conjugated
antisera against M hyorhinis.
$Baltimore Biological Laboratories, Cockeysville,
Md.
Arthritis a d Rheumatism, Vol. 14, No. 2 (March-April 1Sn)
MYCOPUSMA HYORHlNlS
Preparation of the control inoculum. One hundred and nineteen 6- to 8-day-old chick embryos
were given 0.1 ml of broth used in preparation of
the M hyorhinis inoculum. Allantoic amniotic fluid
was collected from viable embryos 5 to 11 days
postinoculation. The pool was negative in mycoplasma broth and on agar, as well as on blood agar,
thioglycollate, Sabouraud chocolate agar and cystine
dextrose blood chocolate agar.
T h e first 5 days postinoculation all animals appeared well. O n the fourth day,
acute and severe illness became abruptly
apparent in the infected herd with fever,
listlessness, and a reluctance to move. T h e
body temperatures averaged 2" C above
those of the control herd. By the fifth day a
frank polyarthritis was seen with swollen
joints, toe-walking, and limping (Fig 1).
Every animal in the group had arthritis by
the middle of the second week. As the
arthritis became more pronounced, the toxic state seemed to recede about two weeks
after inoculation. T h e body temperatures
returned to control levels, never again to
show significant elevations.
I n some animals the arthritis was so
severe that the caretaker had literally to
put the animals on their feet so that they
could reach food and water. At 6 weeks,
mean body weight of the infected animals
was 32 pounds whereas that of the controls
was 54: five months later the controls averaged 280 and the infected herd 155 pounds.
T h e affected joints in any one animal
tended to persist, but there were instances
in which one joint cleared while another
became inflamed. T h e inflammation was
generally symmetrical in its distribution.
Swelling was observed first and most frequently in the ankle: somewhat later wrist
involvement became more prominent. Observations of swelling at the knee or elbow
were made in about a third of the infected
herd. Direct observation of the hips and
shoulders was not possible, but there were
numerous occasions when a limp was apparent without clinically detectable involvement at the more distal sites.
Only 3 infected animals survived beyond
8 weeks. Initially, their disease seemed less
severe, but by the sixth week, they had very
pronounced arthritis. There was slow improvement over the next few weeks: they
became able to move about with less difficulty, but joint swellings were apparent in
all until the time of death or sacrifice.
Attempts to recover synovial fluid in life
were not successful. Lateral and anteriorposterior radiographs of the distal limbs
were made at 8 and 19 weeks. For the most
part the films were free of movement artifact and of excellent quality. T h e films of
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
195
Infectlon of herd. When the swine were about
5 weeks of age, each of one group of animals was
given an intraperitoneal (IP) inoculation of 2 ml of
the M hyorhinis pool. Each animal in the other
group was similarly inoculated with 2 ml of the
control pool. T h e inoculations were camed out on
study Day 0.
Sacrifice
An animal from each group was selected for
sacrifice within a few days of each other. Since a
spontaneous death would compromise the microbiologic findings and since severe, life-threatening
illness was promptly observed, the planned schedule
and random selection was modified to select animals
who were judged to be moribund.
Light anesthesia with intravenous sodium pentobarbital was followed by bleeding and subsequent
sacrifice with a larger dose. The autopsies were
performed immediately with full skin preparation
and aseptic procedures including gown, mask, and
gloves. Using human anatomic designations, the
ankle, knee, hip, wrist, elbow and shoulder of one
side were examined, sampled, and cultured as
described. Subsequently, the abdominal, thoracic
and CNS contents were similarly sampled and
cultured as described. Finally, without sterile precautions, the opposite limb joints were inspected
and sampled for histopathologic preparations only.
Tissues were fixed in 10% buffered formalin and
stained with hematoxylin and eosin for histologic
examination.
R ESU LTS
Clinical
BARDEN & DECKER
the infected, arthritic animals were indis- shaped, erythematous skin lesions appeared
in the control herd on Day 20. Blood
tinguishable from those of the controls.
Clinically characteristic swine erysipelas cultures were positive for Erysifielothrix
with fever, listlessness and large, diamond- insidiosa in 3 of the 7 animals that were
INFECTED
25
* - Socrificed because sick
2eB
36
I
I
18
22
WEEKS POSTIN'JECTION
I
26
310
I
34
Fig 1. Signs and manifestations of arthritis, such as joint swelling, toe walking, limping, and reluctance
to move, were assigned arbitrary numerical values depending upon severity. Values for each day's observations were totaled and the totals are reflected in the height of the black bars above.
1%
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
MYCOPUSMA HYORHlNlS
r
-
14
E
E
a
0
fl
0
12
c
v)
J
-I
W
V
10 -
w
I-
>
0
8
0
-
I
a
2
3
6'
Infected
!d
I
0
I
2
I
6
I
I
I
I
10
14
18
22
WEEKS POSTINJECTION
I
26
I
30
I
34
Fig 2. Mean absolute lymphocyte counts of surviving animals at each time point.
clinically ill. T o e walking, limping, and
hesitancy to move were briefly apparent.
T h e entire herd was given penicillin for
four days with prompt recession of the
findings in the controls and without recurrence. All subsequent efforts to isolate E
insidiosa from either herd were unavailing.
Biochemical and Hematologic Studies
Although there was no significant elevation of the total WBC in the infected herd,
a peripheral blood lymphocytosis was apparent from the third through the twentieth weeks (Fig 2). They also showed a
remarkable rise in total serum proteins.
Most of this was due to a sharp rise in
7-globulins which reached a peak level
within three weeks (Fig 3). T h e high value
was sustained until the twelfth week when
lower values appeared. Thereafter, the infected herd showed a gradual rise paralleling, at a higher level, that of the control
group. T h e ESR rose transiently in the
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
infected swine but, in subsequent months,
remained low despite active polyarthritis.
T h e HCT, SGOT, alkaline phosphatase,
BUN, total serum bilirubin and serum glucose in infected animals was essentially the
same as those of the control swine. No
consistent pattern of response was apparent
on BFT and cold agglutinin testing. spa.
radically positive results appeared in both
herds and were usually followed within a
week or two with a return to negative
values.
Microbiologic
N o mycoplasma was ever isolated from
the nasal cavity of any of our animals,
either from the control or the infected
herds. Regular nasal cultures were stopped
after six consecutive weekly series were
negative.
Blood cultures from every Yorkshire
swine in the infected herd were positive for
M hyorhinis three days postinoculation
197
BARDEN 81 DECKER
r
-
I
I
I
I
OF?
6
I
I
0-0
I
I
10
14
18
22
WEEKS POST INJECT ION
Infected
Control
1
1
1
26
30
34
Fig 3. Mean 7-globulin values of surviving animals at each time point, as determined by serum protein
electrophoresis.
(Table 1). Six weeks later all blood cultures were negative and remained so
thereafter. Weekly bleedings were discontinued after nine weeks but additional
blood cultures were taken on each animal
Table 1. Proportion of M Hyorhinis-Positive
Blood Cultures from Infected Herd
Day of
study
No.
positive
No.
cultured
~
3
10
10
6
17
24
31
38
45
52
60
4
3
0
66
198
0
2
0
0
0
~
10
9
8
7
5
4
5
4
3
3
~~
at 18 weeks and again at the time of
sacrifice. Blood cultures from the controls
were consistently negative for M hyorhinis.
The first two months postinoculation, M
hyorhinis organisms were easily and regularly recovered from each infected animal
at postmortem examination (Table 2).
However, after four months, it became
more difficult. The organism was recovered
from only two of 16 joint cultures from
broth inoculation. No mycoplasma was
ever recovered from a postmortem examination of the controls.
Metabolic inhibition test antibody titers
in swine infected with M hyorhinis began
to rise after one month to a geometric
mean titer greater than 1:lO. Peak titers
were reached around five months postinfection (Fig 4). Individual animal titers within each group did not vary significantly
from one another at any time through the
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
MYCOPLASMA HYORHlNlS
Table 2.
Day of study
Pig number
Recoveries of
4
25
5
24
loo*
104
104
ND
M
Hyorhinis from Infected Herd Postmortem
11
22
18
20
25
31
-
+
31
18
46
19
57
27
114
28
190
33
224
36
ND
ND
ND
ND
ND
-
ND
ND
ND
ND
ND
-
-
ND
-
+
+
ND
+
-
-
ND
ND
+
ND
ND
-
-
+
ND
ND
-
-
ND
ND
ND
-
ND
-
-
ND
ND
-
+
+
ND
+
+
ND
-
-
-
-
ND
103
-
ND
-
-
+D
+
++
+
++
102
-
Abdomen
Peritoneal fluid
Inguinal node
Mesenteric node
Spleen
Liver
+
103
102
+
+
+
+
103
ND
106
+
-
-
-
-
ND
-
-
ND
Thorax
Pleural fluid
Pericardial fluid
Mediastinal node
Lung
Heart muscle
Central nervous system
Cerebrospinal fluid
107
10s
+
+
+
+
+
103
102
+
ND
108
103
104
103
103
+
106
ND
+
-
ND
-
ND
ND
Joints
+
Shoulder
Elbow
Wrist
Hip
Knee
Ankle
N D ND
N D N D +
+
+
lo*
10'
++
-
+
+
+
-
-+
+
++
ND
+
++ N+
++
+ ++ NN DD
+ -N D
--
-
-
10'
-
-
~~
* CFU/ml
on agar and color changing units/ml in broth; all titered in the same r u n
ND = not done;
= positive; - = negative
+
study. The mean titers in the control swine
never exceeded 1:2.
Although there was limited material
available for synovial fluid antibody
studies, preliminary data suggest there is
local antibody production in the joints
(Table 3).
DISCUSSION
The care used in the acquisition of the
young pigs for this study was deliberate; we
wished to inoculate the M hyorhinis into
animals with no earlier contact with the
agent. The organism, often found in the
Arthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
nasal cavity in field herds, was identified in
the nose of several prospective sows. Antibodies to M hyorhinis were found in their
sera. Delivery by hysterectomy and the SPF
isolation were thus deemed necessary and
appear to have been successful. No nasal
isolates were made from the study swine,
no mycoplasma were ever recovered from
the control swine, and antibodies were not
detected in their sera.
The clinical evolution of the acute disease conforms to findings of Roberts et QZ
(3, 4). The rapidly developing polyarthritis persisted as a severe acute episode for
199
BARDEN L DECKER
w Infected
Conlrol
0--0
*
0
Fig 4.
-
2
0
6
10
14
18
22
WEEKS POSTINJECTION
Metabolic inhibition l e s t liters in
Synovial Fluid and Serum
Synovial fluid
Pig Study
no. day
31
28
36
36
25
114
224
224
* Not done
200
30
34
Geometric mean titers of the metabolic inhibition test on surviving animals at each time point.
about two months. The initial period was
followed by some improvement, but clinical evidence of joint disease lasted for eight
months, the duration of the study. The
process seemed to be a steady one, producing chronic disability without evidence of
remission or bursts of greater disease activity.
The microbial involvement of virtually
all joints, the lymphatic system and many
parenchymal tissues as well as the serous
Table 3.
26
Joint
Hip
Hip
Hip
Elbow
Culture
+
ND*
+
-
MIT
titer
Serum
MIT
titer
1:1024
1:1024
1:1024
1:512
1:128
1:64
1/128
1:128
surfaces (Table 2), reflect the widespread
nature of the acute phase of the illness. As
the disease progressed and became chronic,
it was increasingly more difficult to recover
the organism. In the last two animals, after
7 months infection M hyorhinis was recovered from only two sites, both joints. Projection of these findings into still later
phases raises the possibility that culturable
organisms will be spontaneously eliminated, as is seen in other mycoplasmal
diseases (17). However, neither the MIT
titers nor the clinical arthritis could be
regarded as on the wane in the later phases
of this study. More animals through a
longer study would permit assessment of
the persistent joint disease and antibody
evidence of infection.
The absolute lymphocytosis and significant rise in 7-globulin levels suggests that
this mycoplasmal disease may have an
immunologic component more damaging
to its host than the organism itself. The
known half-lives of mammalian immunoArthritis and Rheumatism, Vol. 14, No. 2 (March-April 1971)
MYCOPLASMA HYORHlNlS
globulins and their performance after primary immunization permit speculation
that the initial peak observed was constituted primarily of IgM a n d the later IgG.
Initially, we assumed t h a t the steadily
rising M I T titers were, i n part a t least,
responsible for the failure to recover the
organism. No blood cultures were positive
in animals with titers of 1:64 or greater,
a n d the poor late phase recoveries were
associated with titers in the 1:512 range.
T h a t this ready assumption may not be
t r u e is indicated by the unexpected finding
of synovial fluid titers of 1:1024 i n joints
from which viable organisms could be
recovered.
T h e notable differences between simultaneous synovial fluid a n d serum antibody
levels implies the local production of antibody. I n one of the joints assessed, the fluid
titer exceeded that of serum in the absence
of viable organisms. Greater synovial fluid
titers of antibody to a microrganism has
not, to our knowledge, been reported in
other forms of articular disease.
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS
The authors are indebted to Drs. R. M. Chanock
and D. E. Soghor for advice and assistance with this
work. The expert animal care was directed by Dr.
Walter Kundzins. The technical assistance of Mrs.
Janie Taylor, Mr. I. E. Fishburne, and Mr. Jose R.
Valdesuso is gratefully acknowledged.
12.
REFERENCES
14.
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2. Ferguson RH, Worthington JW: Recent
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3. Roberts ED, Switzer WP, Ramsey FK: T h e
pathology of Alycoplasma hyorhinis arthritis produced experimentally in swine.
Amer J Vet Res 24:19-30, 1963
4. Roberts ED, Switzer WP, Ramsey FK: PaArthritis and Rheumatism, Yol. 14, No. 2 (March-April 1971)
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17.
tllology of the visceral organs of swine
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Ennis RS, Dalgard DW, Willerson JT, et
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Jendrassih L, Graf P: Vereinfachte photometrische methaden zur Bestimmung des
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Hoffman WS: A rapid photoelectric method for the detection of glucose in blood
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201
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