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thesis entitled

INDIRECT IMMUNOFLUORESCENT EXAMINATION OF FROZEN LUNG
SECTIONS FOR DIAGNOSIS OF MYCOPLASMAL PNEUMONIA IN
SWINE WITH MACROSCOPIC AND MICROSCOPIC LESIONS

presented by
HECTOR 0. CHACON

has been accepted towards fulfillment
of the requirements for

M . S . degree in PATHOLOGY

 

 

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Date /} 5

0-7 639

 

 

 

 

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INDIRECT IMMUNOFLUORESCENT EXAMINATION OF FROZEN LUNG
SECTIONS FOR DIAGNOSIS OF MYCOPLASMAL PNEUMONIA IN

SWINE WITH MACROSCOPIC AND MICROSCOPIC LESIONS

BY

Hector 0. Chacon

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1981

 

ABSTRACT
INDIRECT IMMUNOFLUORESCENT EXAMINATION OF FROZEN LUNG

SECTIONS FOR DIAGNOSIS OF MYCOPLASMAL PNEUMONIA IN
SWINE WITH MACROSCOPIC AND MICROSCOPIC LESIONS

BY

Hector 0. Chacon

Thirty pig lungs, selected for presence of macroscopic and/or
microscopic lesions suggestive of mycoplasmal pneumonia in swine, were
studied. Twenty had macroscopic lesions and all 30 had microscopic
lesions. Frozen lung sections, cut to 6 u thickness, were positive on
indirect fluorescent antibody test (IFA) for Mycoplasma hyopneumoniae
in 26 of the 30 pig lungs.

No bacterial culture or any other laboratory tests were done with
the 4 pig lungs IFA negative, and the etiology of the lesions of these
was not determined. The specificity of the IFA test for detecting
M. hyopneumoniae in lung tissue of swine has been established by previous
studies, but it is recommended that further stUdies be done to ascertain
the degree of sensitivity of the test.

The results of this study and those of other investigators reviewed
in this thesis support the use of the IFA test as a relatively simple,
rapid and effective method for detecting M. hyopneumoniae in pig lung
tissue and as an alternative to culturing the organism for etiologic
determination of mycoplasmal pneumonia in swine, suggested on the basis

of macroscopic and/or microscopic lesions observed.

Dedicated with love to my wife, Nancy, for her

constant support and understanding

ii

ACKNOWLEDGEMENTS

I wish to express my sincere thanks to Dr. Rodney Flint Taylor,
my major professor. I also wish to thank Drs. Allan L. Trapp, John
P. Newman, George A. Padgett, and David J. Ellis for serving on my
committee.

A special thank you is extended to Dr. C. H. Armstrong for his

advice and for providing materials essential to the realization of

this study.

iii

TABLE OF CONTENTS
Page
nwmmmmnm. ... ... ... ... ... ... ... ... ... 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . . 3

History . . . . . . . . . . . . . . . . . . . . . . . . . . .
Name of the Disease. . . . . . . . . . . . . . . . . .
Etiologic Identification . . . . . . . . . . . . . . .

The Organism. . . . . . . . . . . . . . . . . . . . . . . . .
Nomenclature . . . . . . . . . . . . . . . . . . . . .
Growth Requirements. . . . . . . . . . . . . . . . . .
Isolation. . . . . . . . . . . . . . . . . . . . . . .
Identification . . . . . . . . . . . . . . . . . . . .
Morphologic Features . . . . . . . . . . . . . . . . .

Epidemiology. . . . . . . . . . . . . . . . . . . . . . . .
Survival of the Organism . . . . . . . . . . . . . . .
Host Range . . . . . . . . . . . . . . . . . . . . . .
Transmission of the Disease. . . . . . . . .». . . . .
Contributory Factors . . . . . . . . . . . . . . . . .
Geographical Distribution. . . . . . . . . . . . . . .

Clinical Picture. . . . . . . . . . . . . . . . . . . . . . .

Pathology . . . . . . . . . . . . . . . . . . . . . . . . . .
Macroscopic Lesions. . . . . . . . . . . . . . . . . .
Histopathology . . . . . . . . . . . . . . . . . . . .

Serologic Procedures. . . . . . . . . . . . . . . . . . . . .
Complement Fixation Test (CFT) . . . . . . . . . . . .
Latex Agglutination Tube Test. . . . . . . . . . . . .
Indirect (Passive) Hemagglutination (IHA). . . . . . .
Fluorescent Staining Antibody. . . . . . . . . . . . .

Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . .
Nonspecific Diagnosis. . . . . . . . . . . . . . . . .
Specific Diagnosis . . . . . . . . . . . . . . . . . .
Differential Diagnosis . . . . . . . . . . . . . . . .

\OLOkOKOKOlemONONO‘Ib-wa

mumlandietanAIAIAIAIAiAiata
F'OIO\D~J~JO\U1UIO)C>C>O 0

MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . . 23

Lung Specimens. . . . . . . . . . . . . . . . . . . . . . . . 23
Antiserum-Conjugate-Controls. . . . . . . . . . . . . . . . . 7?
Macroscopic Lung Evaluation . . . . . . . . . . . . . . . . . 24
Histopathology. . . . . . . . . . . . . . . . . . . . . . . . 25
Tissues for Immunofluorescence Staining . . . . . . . . . . . 25
Indirect Immunofluorescent Staining Antibody (IFA). . . . . . 26

iv

Page
RESULTS 0 O C O O O O O O O O O O O O I O O 0 O O O O O O O O O O O O 27

Macroscopic Lesions . . . . . . . . . . . . . . . . . . . . . 27
Histopathology. . . . . . . . . . . . . . . . . . . . . . . . 27
Pig Lungs with Macroscopic and Microscopic Lesions . . 27
Pig Lungs with Only Microscopic Lesions. . . . . . . . 34
Summary of Histopathology. . . . . . . . . . . . . . . 36
Indirect Immunofluorescent (IFA) Staining Results . . . . . . 36

DISCUSSION 0 O O. O O O O O 0 O O O O O O O O O O O O 0 O O 0 O O O O 47
BIBLImmHY O O C O O O O O O O O O O O O O O O O O O O O O O O O O 50
VITA . . . .

APPENDIX . .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 56

LIST OF TABLES
Table Page

1 Number of pig lungs collected and lesions . . . . . . . . . . 24

2 Grading scheme of pig lung lesions according to percent

average of consolidated right and left anteroventral lung

lobe fields 0 O O O 0 O O 0 O O O O O O O O O 0 O O 0 O O I O 25
3 Grading scheme of pig lung lesions according to percent

average of consolidated right and left anteroventral lung
lobe fields (number of pig lungs by grade). . . . . . . . . . 28

4 Summary of morphologic diagnosis and age of each study pig;
lungs with macroscopic and microscopic lesions. . . . . . . . 32

5 Summary of morphologic diagnosis and age of each study pig;
pig lungs showing only microscopic lesions. . . . . . . . . . 34

6 Summary of indirect FA test in pig lungs with macroscopic

and microscopic lesions and age of each study pig . . . . . . 44
7 Summary of indirect fluorescent antibody test (IFA) in pig

lungs which had no macroscopic lesions but did have micro-

scopic lesions, and age of each study pig . . . . . . . . . . 45

vi

Figure

10

LIST OF FIGURES

Pneumonic pig lung.
cardiac lobes . . . . . . .

Pneumonic pig lung.
lobes O O I O C O O O O O O

Pneumonic pig lung.
lobes.

healthy lung. . . . . . . .

Photomicrograph of pig lung.

Notice accumulations of

O

Lobular distribution of the lesions.
separation between affected lung and normal appearing

O

O

Bilateral and symmetrical consoli-
dation of the anteroventral parts of the apical and

lymphocytes in peribronchiolar and perivascular spaces.

Photomicrograph of pig lung.

Notice accumulations of

Consolidated right apical and cardiac

Consolidated left apical and cardiac
Clear-cut

O

lymphocytes in peribronchiolar spaces and alveolar lumens .

Photomicrograph of pig lung.
lymphocytes.
FibrOblaSts O O O O O O O O

Photomicrograph of pig lung.

Peribronchiolar cuffing with
Peribronchiolar lymphoid hyperplasia.

Debris, lymphocytes, and

neutrophils in bronchiolar lumen.

Photomicrograph of pig lung.

Notice infiltration of

O

lymphocytes in alveolar walls and bronchiolar spaces. . . .

Photomicrograph of pig lung.
of lymphocytes. . . . . . .

Photomicrograph of pig lung.

spaces.

Peribronchiolar accumulations

Notice accumulations of
lymphocytes in peribrOnchialéperibronchiolar-perivascular

vii

Peribronchial-peribronchiolar lymphoid hyperplasia .

Page

30

3O

31

38

38

4O

4O

42

42

43

INTRODUCTION

Diseases are limiting factors for pig production. Infectious,
nutritional, toxic, genetic, and environmental factors play a role in
the diseases of pigs. As for the infectious diseases, the etiological
agents are bacterial, viral, parasitic, fungal, and mycoplasmal.

Such diseases have been studied with microbiologic, clinicopathologic,

and epidemiologic methods for purposes of treatment, control, and/or
eradication. Swine mycoplasmosis exhibits a varied clinicopathologic
picture as well as distinctive epidemiologic behavior. Swine mycoplasmal
pneumonia is caused by Mycoplasma hgopneumoniae. It may act with one

or multiple secondary disease-producing agents, and it affects swine
regardless of breed, sex, and age. It is a chronic disease with high
morbidity and high mortality in complicated forms. Swine mycoplasmosis

is important economically because affected animals usually grow more
slowly.

In mycoplasmal pneumonia in swine, there has been an accumulation
of knowledge on the pathologic aspects of the disease in the last 50
years. In the last 15 years, there has been etiologic definition.
Diagnosis of mycoplasmal pneumonia in swine still remains a complex
matter because of "common clinico-pathological features" shared with
other swine diseases.

Relatively recently, serologic techniques, epidemiologic observa-

tions, and pathologic studies are helping in the diagnosis of swine

2
pneumonia caused by M. hyopneumoniae. The fluorescent antibody staining
technique has proven to be a useful supportive diagnostic procedure.

On the basis of the aforementioned concepts, this study was con-
ducted to attempt to demonstrate the effectiveness of the indirect
fluorescent antibody test as a supportive diagnostic procedure of
mycoplasmal pneumonia of swine, given macroscopic and/or microscopic

lesions suggestive of the disease.

LITERATURE REVIEW

History

Name of the Disease

 

A chronic respiratory disease of swine was reported by Kobe (1933,
1934) in Germany, which was known as "Ferkelgrippe." In Northern
Ireland, Lamont (1938) reported a disease in swine with similar clinico-
pathologic characteristics to those described by Kobe, and he named it
pig influenza. Several different names were given over the years by
several investigators in different countries to‘a disease entity having
clinicopathologic characteristics of the diseases described by Kobe
(1933, 1934) and Lamont (1938). Among these names were: 1) infectious
pneumonia of pigs (Pullar, 1948; Gulrajani and Beveridge, 1952),
2) swine enzootic pneumonia (Wesslen and Lannek, 1954; Lannek and Wesslen,
1957); 3) virus pneumonia of pigs (Betts, 1952; Beveridge, 1953; Betts
and Beveridge, 1952). Despite the different names given to this "respira-
tory disease in swine", there was overall agreement on the descriptions
of the disease (Switzer and Ross, 1975). In 1952, a possible relationship
among the above-mentioned diseases was considered in a review written

by Lamont (1952).

Etiologic Identification
In their studies, Kobe (1933) and Lamont (1938) reproduced the

disease experimentally by inoculating healthy pigs intranasally with

4
pneumonnc lung suspensions, and they each concluded that a filterable
virus was the primary etiologic agent, with the participation of
HaemOphilus influenza suis as a secondary invader (Kobe, 1933, 1934;
Lamont, 1938). The latter organism was previously identified by Lewis
and Shope in their studies of swine influenza and who gave it its name
(Lewis and Shope, 1931).

A clinicopathologic entity was produced by intranasal inoculations
of H. influenza suis plus one or more other pathogenic microorganisms
considered as secondary invaders, such as Bacillus pyocyaneus (synonym
for Pseudomonas aeruginosa). Bacillus pyocyaneus and Salmonella suis
infections also showed clinical similarities with pig influenza (Lamont,
1938). From work done by Betts (1952), Betts and Beveridge (1952),
and Beveridge (1953), it was presumed that the etiologic agent of this
chronic pig pneumonia was a filterable virus. The presumption that a
filterable virus, distinct from swine influenza virus, was the etiologic
agent of an "infectious pneumonia of pigs" was also held by Pullar
(1948). From their work on experimental transmission of the disease,
Gulrajani and Beveridge concluded that "infectious pneumonia of pigs" '
was caused by a viral agent (Gulrajani and Beveridge, 1951). The assump-
tion that the causative agent of "chronic pneumonia of swine" was a viral
agent was reinforced by the fact that pneumonic lung suspensions, passed
through bacteria-retaining filters, proved infectious to inoculated
healthy swine, and this view persisted until 1957, when Betts and
Campbell (1956) demonstrated that enzootic pneumonia of swine could be
prevented with administration of tetracyclines. The possibility that
the etiologic agentof pig enzootic pneumonia could be a pleuropneumonia-
like organism (PPLO) was expressed by Wesslen and Lannek (1954), and

pleuropneumonia-like organisms associated with pneumonia in swine were

 

 

5
also reported by Carter and Schroder (1955). Using pneumonic lung
suspensions, a cytopathic effect was observed in "pig lung monolayer
cell culture" and pleomorphic organisms were seen by light microscope
(Goodwin and Whittlestone, 1963). These reported pleomorphic organisms
were propagated in pig lung cell culture, which cells had been killed
by boiling (Goodwin and Whittlestone, 1964). Pneumonia in swine, pro-
duced by the inoculation of Mycoplasma cultured in lifeless media, was
reported by Goodwin and Whittlestone (1965), and the name Mycoplasma
suipneumoniae was proposed for the pathogenic microorganism. In the
U.S., Mare and Switzer (1965, 1966a,b) grew and propagated the causative
agent of a chronic pneumonia in pigs, using artificial media, and then
characterized it as a Mycoplasma and named it Mycoplasma hyopneumoniae
(Mare and Switzer, 1965). Results like those reported by Goodwin and
Whittlestone (1965) in England and Mare and Switzer (1965) in the U.S.
were also reported by L'Ecuyer (1969) in Canada. The results of the
work done by Goodwin and Whittlestone in pigs were reported shortly
after those of Mare and Switzer. In 1967, M. hyopneumoniae and M.
suipneumoniae organisms were compared by Goodwin et a1. (Goodwin,
Pomeroy and Whittlestone, 1967), and they concluded that both were the
same agent. Since the designation of M. hyopneumoniae had chronological
priority over M. suipneumoniae, the former name has been used to designate
the causal agent of a chronic pneumonia in swine. Over the years, M.
hyopneumoniae isolations in cases of chronic pneumonia in swine have
been reported in other countries: Denmark (Friis, 1971, 1975; Meyling.
1971) and Australia (Furlong et a1., 1975; Etheridge et a1., 1979).
Reports of M. hyopneumoniae isolations continue to fill the literature.
In this thesis chronic pneumonia of swine caused by M. hyopneumoniae

will be referred to as mycoplasmal pneumonia of swine.

6

The Organism

 

Nomenclature

 

Mycoplasma sp. are placed in one order, Mycoplasmatales, one family,
Mycoplasmataceae, and one genus, Mycoplasma, all in the class Mollicutes
(Buxton and Fraser, 1971). These pleomorphic organisms are distinguished
as species on the basis of serologic reactions, and about 25 to 30

different Mycoplasma sp. have been recognized.

Growth Requirements

The Mycoplasma sp. are fastidious organisms. For growth, complex
media containing meat infusion broth, Bacto-brain heart infusion,
Bacto-PPLO without crystal violet, peptones, sodium chloride, yeast
extract, some other chemicals, and porcine and/or horse serum are needed.

The optimum temperature for growth is 37 C.
Isolation

Isolation from 1ung_in solid media. “In experimentally infected
pigs{ direct isolation of M. hyopneumoniae colonies from bacteriologic
lung culture on solid media has been positive in a few cases, but for
practical purposes of routine diagnosis this technique is not considered

satisfactory (Whittlestone, 1973).

Isolation in tissue culture. Isolates of pleomorphic organisms
were first obtained by tissue culture methods. A cytopathic effect has
been observed in both pig lung monolayer cell culture and pig testicle

cell culture (Wesslen and Lannek, 1954; Goodwin and Whittlestone, 1963).

Isolation in liquid media. The first reported lifeless media to be

used for bacteriologic culture of M. hyopneumoniae were: 1) a tissue

 

7
culture feeding fluid and 2) broth with dead pig lung cell culture
(Goodwin and Whittlestone, 1965; Mare and Switzer, 1965). FOllowing

the successful results of the isolation attempts above reported, other
media have been developed for bacteriologic culture of M. hyopneumoniae
or other Mycoplasma sp. of porcine origin, and details and methods for
preparation of these media have been described elsewhere (Switzer, 1972;

Friis, 1971, 1975; Goodwin, 1976).

Identification

 

The Mycoplasma sp. organisms are isolated in broth media. This
isolate is then grown in colony form on a filter membrane and can then
be identified by means of an indirect immunofluorescent test. The iso-
lation and identification process requires about 2 weeks (Armstrong,
1980). Other means for identification of isolates are metabolism inhi-

bition and growth inhibition (Whittlestone, 1973).

Morphologic Features

 

Usually, M. byopneumoniae colonies may be detected when subcultured
on solid media after 3 days of incubation, having a diameter of 20 to
100 um and reaching 0.5 mm in diameter after 7 to 10 days of incubation.
The colonies commonly are convex with a granular surface and with no
observable central portion burrowing into the media (Whittlestone, 1973).
The morphological appearance varies from a) coccoid to b) ring-like
triangles to c) rod-like or irregular shape (Whittlestone, 1973). The
variety of morphologic structures which have been observed and described
for M. hyopneumoniae seem also to be influenced by the method of growth

and preparation used, as below.

 

8

Lung_tiSSue. The organism manifests itself morphologically as

 

rings, cocci, triangles, and other less common forms. The observed
size is about 0.5 pm in diameter for ring-like organisms, about 0.2

to 0.5 um in diameter for coccoid-like organisms, and about 1.0 pm in
diameter for triangular organisms. The cilial region of the bronchial
epithelial cells, as well as between the cilia, were the sites where

the organism was found most easily (Whittlestone, 1973).

Broth culture. Circular colonies, mainly coccoid-like, reaching

 

an average diameter of 10 to 25 um, have been seen growing over the
surface of glass coverslips, according to Goodwin and Whittlestone

(1966, cited by Whittlestone, 1973).

Tissue culture. Coccoid— and ring-like forms were usually seen
in tissue culture, frequently in large groups and at times extracellu-

larly located (Goodwin and Whittlestone, 1963).

Solid media. On agar medium, many Species of Mycoplasma have a
morphologic appearance which is referred to as "fried egg" colonies,
but M. hyopneumoniae usually do not form "fried egg" colonies.

Structurally, Mycoplasma sp. lack a cell wall, as do the L-forms
of bacteria. However, there are basic and substantial differences,
such as: l) L-form.bacteria, as well as occurring naturally, can also
be induced by laboratory methods; 2) most L-form bacteria revert to
their parent bacteria; 3) L-form bacteria have the nutritional and bio-
chemical properties of their progenitors. The pleomorphism observed
in Mycoplasma sp. is due to a great extent to the shape imposed by the
physical environment and facilitated by the absence of a cell wall

(Madden et a1., 1967).

 

'9

Epidemiology

Survival of the Organism

In pneumonic lung tissue, M. hyopneumoniae remains infective at
-30 C for up to 20 months (Whittlestone, 1958), at 4 C for 4 days,

at 20 C for 1 day, and at 37 C for 4 hours (Whittlestone, 1958).

Host Range

Swine are the only animals known to be susceptible to M. hyopneu-
moniae infection. Experimental transmission attempts to laboratory
animals such as ferrets and mice (Goodwin and Whittlestone, 1963, 1964)

and mice (Wesslen and Lannek, 1954) have been unsuccessful.

Transmission of the Disease

The disease may be transmitted over short distances by aerosol from
infected to susceptible swine; in the natural environment the organism
appears to be short-lived (Switzer and Farrington, 1977). Pneumonia is
also produced when cultures of M. hyopneumoniae are inoculated into
healthy swine. Lung suspensions from these swine inoculated into healthy

pigs also produce pneumonia (Pattison, 1956).

Contributory Factors

Consideration has been given to a multiple factor disease criterion,
in which other factors in addition to the infective agent play an
:Unportant role. Bacterial agents, parasites, dust, humidity, or irritant
gases may be associated with mycoplasmal pneumonia in swine. These factors
<iisrupt the lung's normal functioning, lowering the natural resistance to
iJrfection (Jericho, l968),and contribute to the severity of the disease
anud characteristics of gross pathology (Schofield, 1956). Mycoplasma

Inflqpneumoniae itself is known to be immunosuppressive (Adegboye, 1978).

 

10
Geographical Distribution
The disease is considered to be of worldwide distribution, based on
descriptions of the clinical signs, macroscopic and histologic lesions,
as well as isolation of the M. hyopneumoniae agent and epidemiologic

observations of the disease.

Clinical Picture

The clinical course of mycoplasmal pneumonia of swine is chronic
and relatively mild in uncomplicated cases. Swine grow slowly and cough
with no increased body temperature and show dyspnea on rare occasions.
Progressive involvement with respiratory signs can be recognized through-
out all age groups in large infected herds which are intensely managed.
Adult animals can be severely affected. Although there is a high mor-
bidity rate, the case fatality rate is generally low (Switzer and Ross,
1975; Switzer and Farrington, 1977). Complicated forms of the disease
are frequently observed if the swine are exposed to contributory patho-

genic factors, and in such situations the mortality rate can be higher.

Pathology

Macroscgpic Lesions

 

At necropsy of swine which died of mycoplasmal pneumonia, the
most common macroscopic lesions found in the lungs are described
as circumscribed, plum-colored, grayish, consolidated areas. Primarily
affected are the anteroventral parts of the anterior lung lobes (the
right cardiac, the right apical, the left cardiac, the left apical, and
the intermediate lung lobes), all of which show about the same incidence
of lesions with a symmetrical tendency. The resulting pneumonia is

catarrhal in type and of lobular distribution. If a pneumonic lung is

 

ll
incised, the cut surface appears smooth, homogeneous, and wet, and a
clear or mucoid exudate can be expressed by gently squeezing the central
bronchiole. The exudate can be yellowish or opaque, suggesting a
secondary bacterial infection (Schofield, 1956; Switzer and Ross, 1975;
Jericho, 1968). Irregularly distributed emphysematous lobules, atelec-
tatic areas and grayish consolidation, necrosis and abscessation caused
by secondary bacterial infection provide a colorful aspect to the macro-
scopic lesions.

Descriptions of macroscopic lung lesions similar to those mentioned
above were made by Lamont (1938), who also mentioned enlarged pulmonary
lymph nodes, fibrinous pleuritis, pericarditis, and epicarditis. This
author affirmed that on the basis of macroscopic lung lesions, a diagnosis
of swine influenza, as the disease was then known, could be made. Through-
out the years, the lung lesions of both field and experimental cases
have been observed and described by workers in different countries.
These workers are basically in agreement in their descriptions of macro-
scopic appearance of the lesions as enlarged, edematous pulmonary lymph
nodes, grayish consolidation, and pulmonary edema (Kobe, 1934; Pullar,
1948; Pattison, 1956; Schofield, 1956; Switzer and Ross, 1975), with
complete healing if recovery occurred.

In experimental cases of mycoplasmal pneumonia of swine, appearance
of macroscopic lung lesions postinoculation, as reported by Pattison
(1956), has been as follows: at 11 days, "greyish-pink consolidated
areas at the tip of the right and left apical lobes and over about one-
third of the left cardiac lobe"; at 18 days, progressive consolidation;
at 33 days, "consolidation of the entire right apical and left cardiac

lobes and patchy consolidation on the anterior border of the diaphragmatic

lobe."

12

From reports by Whittlestone (1973), macroscopic lung lesions could
sometimes be detected at 7 to 10 days postinoculation and practically
all infected pigs by 6 weeks postinoculation had moderately extensive
pneumonic gross lesions.

Macroscopic lung lesions were observed by L'Ecuyer and Boulanger
(1970) in only 2 of 10 pigs killed between 7 and 21 days postinoculation,
and those 2 were at 12 and 20 days postinoculation, respectively. Of
the 12 remaining pigs, killed between the 25th and 49th postinoculation
days, all except 1 showed from slight to extensive gross lesions.

Other workers (Livingston et a1., 1972) have reported that gross
lesions became evident at the 14th day postinoculation and developed
maximum severity between the let and 28th days postinoculation and
then regressed.

Macroscopic lung lesions were observed (Etheridge and Lloyd, 1980)
in experimentally infected pigs 4 to 6 weeks postinoculation, and the
same authors observed macroscopic lung lesions at 4 to 10 weeks post-
exposure in pigs infected by contact with experimentally infected pigs.

In uncomplicated field cases, as well as in experimental cases of
the disease, the macroscopic lung lesions which appear similar persist
for long periods of time. Market weight swine are those which show the
higher incidence, according to reports in the literature (Goodwin and
Whittlestone, 1960; Schofield, 1956; Switzer and Ross, 1975; Switzer
and Farrington, 1977). The incidence of chronic lesions observed is
less in slaughter sows than in butcher-weight swine, which has been
interpreted as an age-clearing condition (Switzer and Ross, 1975).
Macroscopic lung lesions similar to those described for mycoplasmal

pneumonia in swine can also be observed in other respiratory diseases

 

l3
(Pattison, 1956; Whittlestone, 1957; Goodwin and Whittlestone, 1960;

Switzer and Ross, 1975).

Histopathology

In natural cases of the disease, histopathologic descriptions have
been made as follows (Pattison, 1956): slight edema and cellularity
increase in the interalveolar spaces, alveolar lumens filled with a few
large mononuclear cells and with increased edema and cellularity, and
extensive lymphoid hyperplasia, mainly of peribronchiolar and perivascular
distribution. The dense accumulations of mononuclear inflammatory cells
obliterate the alveolar tissue. Substantial amounts of cellular debris
fill the bronchi and bronchiolar lumens. Hyperplastic bronchiolar
epithelium, constricted lumens of bronchi, and bronchiolitis in some
cases were seen. Lymphoid hyperplasia as well as collapse of lobular
tissue were also observed. The above histologic changes described were
those corresponding to consolidated lung tissue (Pattison, 1956).

Plasma cells, a few histiocytes, and neutrophils have also been observed
in the alveolar lumens (Schofield, 1956). Peribronchiolar and peri-
vascular accumulations of inflammatory mononuclear cells, as described
above for the natural cases of the disease, can also be caused by other
pathogenic agents, such as bacteria, parasites, virus, dust, humidity,
or any particulate matter (Jericho, 1968, 1977; Switzer and Ross, 1975;
Whittlestone, 1973).

In field cases of the disease, histologic lung lesions which have
been observed include slight peribronchiolar accumulations of lymphocytes
as well as interalveolar tissue infiltrated by macrophages throughout
the lung lobes. These have been regarded as nonspecific histologic

lesions and frequently have been reproduced by experimental inoculations

14

of fluids (Pattison, 1956; Jericho, 1968; Whittlestone, 1973; Switzer
and Ross, 1975) and are also induced by dust, irritant gases, or any
other particulate foreign matter (Jericho, 1968; Switzer and Ross, 1975).

In pigs experimentally infected with M. hyopneumoniae organisms,
the early histologic lung lesions observed have been as follows:
1) At 7 days postinoculation, there was peribronchiolar and perivascular
lymphocytic accumulation and increased cellularity in the interalveolar
spaces. Neutrophils were rare and there was a very slight bronchiolar
exudate (Pattison, 1956). 2) At 11 days postinoculation, there was
collapse of lobules and increased accumulation of lymphocytes. The
bronchiolar epithelium had a hyperplastic appearance. There were neu-
trophils, small round cells, and exudate in some bronchioles (Pattison,
1956). 3) At 33 days postinoculation there were extensive lymphoid
hyperplasia, accumulation of mononuclear cells in the alveolar walls,
and a few large mononuclear cells with slight edema in the alveolar
spaces. Progressive lymphoid hyperplasia related to bronchi and bron-
chioles led to obliteration of alveolar tissue and dense accumulations
of lymphocytes in the peribronchial and peribronchiolar spaces. The
lumens of the bronchioles often appeared very constricted. It was also
seen that lymphoid hyperplasia could occur in the absence of alveolar
involvement. The bronchiolar epithelium had a hyperplastic appearance
(Pattison, 1956). 4) From 17 to 24 days postinoculation, progressive
lymphoreticular hyperplasia, perivascular accumulations of mononuclear
inflanmatory cells, and development of alveolar cell pneumonia occurred
(Whittlestone, 1973). 5) From 69 to 269 days postinoculation, the
bronchiolar tree appeared surrounded by lymphoreticular nodes and the

ciliate bronchiolar epithelium was regenerated (Whittlestone, 1973).

 

15

As for the presence of M. hyopneumoniae organisms associated with
the surface of the bronchiolar epithelium, it has been reported in
large numbers in the period from 7 to 42 days postinoculation
(Livingston, Stair, Underdahl, and Mebus, 1972; Whittlestone, 1973;
Kobisch, Tillon, and Vannier, 1978), and low numbers have been detected
up to 262 days (37 weeks) postinoculation in experimentally infected
swine (Whittlestone, 1973). As for the lung pathologic examination,
it has been observed that in field conditions many pigs do not develop
macroscopic lesions but will show microscopic lesions, and swine with
macroscopic and histologic lesions have been observed coming from areas

which are considered "swine lung normal areas" (Switzer and Ross, 1975).

Serologic Procedures
As an aid for the diagnosis of mycoplasmal pneumonia in swine, sero-

logic procedures which have been used include the following.

ggmplement Fixation Test(CFT)

In pigs experimentally infected by inoculation of M. hyopneumoniae,
serum antibodies have been detected by the CFT at 3 to 6 weeks post-
exposure (Switzer and Ross, 1975; Slavik and Switzer, 1979; Etheridge,
1980), and such antibodies persisted for up to 15 weeks postexposure
(Slavik and Switzer, 1979) and also for up to 35 weeks postexposure, as
reported by Etheridge (1980).

In swine infected by contact, CFT serum antibodies have been detected
at 8 to 15 weeks postexposure, while Etheridge (1980) had det-ctsd CFT
serum antibodies at 4 to 40 days postcontact, and such antibodies per-
sisted for up to 35 weeks postcontact.

In field conditions, swine infected with a virulent strain of M.

.hyopneumoniae exhibited clinical signs of pneumonia and showed positive

 

16

titers to the CFT, while in separate observations pigs infected with a
less virulent strain of M. hyopneumoniae usually were CFT negative.
In both instances, the swine Were 8 to 10 weeks of age. It has also
been observed that swine infected with mild strains of M. hyopneumoniae
usually became serologically positive by about 16 weeks of age (Switzer
and Farrington, 1977).

In normal conventionally infected herds, a peak incidence of sero-
positive animals (60 to 65%) in the 6 month to 1 year age range has
been reported, and as the breeding stock gets older, the incidence of
seropositive animals declines to an expected 20 to 25% in animals over
30 months of age. But seronegative animals may become seropositive
during periods of stress (Switzer and Ross, 1975; Switzer and Farrington,
1979). The presence of CFT serum antibodies has been correlated with
observations of gross and microscopic lesions. The CFT titers are still
present when the gross lesions have disappeared. When the CFT titer
converts to negative, microscopic lesions can still be detected (Switzer

and Farrington, 1977; McKean, Andrews, and Farrington, 1979).

Latex Agglutination Tube Test

With this test, in experimentally infected swine (by inoculation with
M. hyopneumoniae), serum antibodies have been detected as early as 2 to
3 weeks postinoculation and for up to 48 weeks postinoculation. The anti-
body titers persist even after the microscopic lung lesions disappear
(Slavik and Switzer, 1979; Switzer and Farrington, 1977). In pigs which

have been infected by contact, positive titers to the test are detected

at 4 to 12 weeks postcontact.

 

l7
Indirect (Passive) Hemagglutination (IHA)

With this test, in experimentally infected swine, high titers of
serum antibodies have been detected 5 to 7 weeks postinoculation and
through a period corresponding to the healing of the macroscopic lung
lesions (about 85 days postinoculation) (Kobisch, Tillon, and Vannier,

1978).

Fluorescent Staining Antibody

 

This technique has been reported as a rapid and reliable method,
which has been applied to smears of broth culture, to infected pig
testicle cell culture, and to frozen sections of consolidated swine
lungs. In the latter case, the pig lungs tested were from animals that
died from experimentally produced mycoplasmal pneumonia or were field
cases of the disease. Fluorescence has also been demonstrated in
mycoplasmas of porcine origin, grown in colony form on a filter membrane.
The fluorescent antibody has proven to be species specific and has been
suggested as an alternative to the bacteriologic culture for isolation
and identification of porcine Mycoplasma sp., a difficult and time-
consuming process (Armstrong, 1980; Kobisch, Tillon, and Vannier, 1978;

L'Ecuyer and Boulanger, 1970; Meyling, 1971; Stone, 1976).

Direct FA. The direct fluorescent staining antibody method was used
for identification of M. hyopneumoniae colonies, grown in lifeless media
as well as sections of consolidated pig lungs, with results reported as
satisfactory (L'Ecuyer and Boulanger. 1970,: Meyling, 1971
Tillon, and Vannier, 1978). Porcine and/or rabbit globulins were used
to perform the direct FA, and porcine globulins appeared more satisfactory

to some workers (L'Ecuyer and Boulanger, 1970; Dobisch, Tillon, and

18
Vannier, 1978), while rabbit globulins appeared satisfactory to others

(Meyling, 1970).

Indirect FA. Mycoplasma sp. of porcine origin grown in colony form
on filter membrane has been identified by indirect fluorescent staining
antibody method. This method has also been used to identify the myco-
plasmal organisms in pig lung showing macroscopic and/or microscopic
lesions. Rabbit globulins and goat-antirabbit globulins have been used
and satisfactory results reported, which permits shortening the period

of time for identification of porcine Mycoplasma sp. (Armstrong, 1980).

FA relatiogship with other conditions. In early stages of myco—
plasmal pneumonia in swine, the results of the direct FA test have been
negative. Convincing positive results Were seen at the 25th day post-
inoculation and thereafter (L'Ecuyer and Boulanger, 1970). Reasons for
this outcome could be due:

1) to the difficulty in obtaining representative sections
of lung, when the areas of pulmonary consolidation are
very limited,

2) to an insufficient amount of antigen being present to

allow visualization, and

to washing out of the antigen from the epithelium of the

air passages during the staining procedure.

3

v

(L'Ecuyer and Boulanger, 1970)
Other workers have reported positive results with the direct FA as
early as 14 days postinoculation of experimentally infected pigs with
mycoplasmal pneumonia (Livingston, Stair, Underdahl, and Mebus, 1972)
and have added that Mycoplasma sp. organisms can be identified in thin
sections of frozen lungs in both experimental and field cases which

showed no clinical signs or macroscopic lung lesions (Kobisch, Tillon,

and Vannier, 1978).

 

19

In pneumonic pig lungs, Mycoplasma hyorrhinis has been isolated
while M. hyopneumoniae has been direct FA positive and culture negative.
Such results have been interpreted as "the Mycoplasma hgorrhinis
blocking and out-growing Mycoplasma hyopneumoniae" (Meyling, 1971).

In chronic cases of mycoplasmal pneumonia in swine, weak or negative
direct FA results have been observed, and they have been attributed to
the action of local antibody (Meyling, 1971; Kobisch, Tillon, and Vannier,
1978). As for the indirect FA, it correlates well with the findings of
macroscopic and/or microscopic lung lesions (Armstrong, 1980).

In pneumonic swine lung, M. hyopneumoniae positive to FA staining,
fluorescent organisms are clearly visible over the surface of the airways

when observed with the fluorescent microscope.

Dia nosis

Nonspecific Diagnosis

Clinical signs and macroscopic lesions. More than one disease can

produce similar clinical signs and macroscopic pathologic changes in pig
lungs (Pattison, 1956; Whittlestone, 1957, 1973; Switzer and Ross,
1975). Similar macroscopic lung lesions produced by different diseases
exhibit histopathologic differences (Pattison, 1956; Whittlestone,
1957, 1973; Goodwin and Whittlestone, 1960; Switzer and Ross, 1975;
Jericho, 1968).

In field conditions a variety of bacteria such as Pasteurella
multocida, P. haemolytica, Bordetella bronchiseptica, Klebsiella pneu-
moniae, and Corynebacterium pyogenes may be associated with mycoplasmal

pneumonia in swine, contributing to the severity of the disease and type

of gross pathology. Lungworms, swine influenza virus, and pseudorabies

 

 

 

20
can also complicate the disease picture, masking the primary changes

(Whittlestone, 1957, 1973; Jericho, 1977; Switzer and Ross, 1975).

Specific Diagnosis

Respiratory disease in swine, suggestive of mycoplasmal pneumonia
on histologic examination and consideration of clinicopathologic and
epidemiologic data, could be confirmed to be mycoplasmal pneumonia only
by recovery of the specific organism from the lung. Isolation and
identification of the pathogenic agent of mycoplasmal pneumonia in
swine is a time-consuming process, not suited for purposes of routine

diagnosis.

Serologic procedures. The complement fixation test could be
helpful if used as a predictor of macroscopic lung lesions, substituting
or supplementing the slaughter inspection for a herd diagnosis (Switzer
and Ross, 1975; Switzer and Farrington, 1977; McKean, Andrews, and
Farrington, 1979). With regard to the latex agglutination tube test
(LAT), with LAT serum antibody titers persisting even after the macro-
scopic lung lesions and CF serum antibodies disappearing, this serologic
procedure could be used as a predictor of microscopic lesions. In such
a way, it would be useful in determining or detecting chronic infected

herds (Switzer and Farrington, 1977; Slavik and Switzer, 1979).

Fluorescent antibody staining. Identification of M. hyopneumoniae

isolates in filter membrane (Armstrong, 1980) or in frozen sections of
consolidated lungs, smears of broth culture and bronchial exudate
(Armstrong, 1980; L'Ecuyer and Boulanger, 1970; Meyling, 1971) by
visualization of the organism in the lung airways makes the FA a suitable

supportive diagnostic procedure for diagnosis of mycoplasmal pneumonia

in swine.

 

21

Differential Diagnosis

Swine influenza. For this pathologic condition there is sudden

 

onset, severe clinical signs, pyrexia, anorexia, painful coughing and
sneezing, and rapid loss of weight. Pathologic changes observed are
pulmonary edema, diffuse acute inflammation, consolidated cranial lung
lobes, and resolution of lesions in 2 to 3 weeks in uncomplicated cases.
Microscopically, bronchial exudate, focal necrosis and degeneration of
bronchial epithelium, peribronchial and perivascular inflammatory mono-
nuclear cell infiltration, polymorphonuclear leukocytes, and focal
coagulative necrosis are observed (Shope, 1931; Whittlestone, 1957).
Isolation of swine influenza virus or demonstration of specific serum
antibodies are sine qua non for diagnosis of swine influenza. Myco-
plasma hyopneumoniae isolation by culture or visualization by FA staining
in frozen sections of consolidated lung tissue will help in a differen-

tiation of the disease.

Lungworms--pneumonia. Clinical signs described for this patho-
logic condition usually are: anorexia, constipation, diarrhea, emaciation,
and coughing. On postmortem examination, areas of emphysema are observed
in the diaphragmatic lung lobes, at the tips or free borders of these
lung lobes; parasites can be found in the bronchiolar lumens. Micro—
scopically, the lung lesions show hypertrophy of smooth muscle, lymphoid
hyperplasia, giant cells and eosin0phils, as well as lungworm eggs in

the alveoli.

Pasteurellosis. In severe cases of this disease, there is fever,
mucopurulent nasal discharge, anorexia, and depression. Exudative
bronchopneumonia of lobular distribution is observed, as well as grayish

lung consolidation and edema. Pleura and peritoneum occasionally show

22
serofibrinous inflammation. Microscopically, lymphocytes, macrophages,
and neutrophils can be seen around the peribronchial and perivascular
spaces. Neutrophils accumulate also in the alveolar spaces. Pasteurella
multocida is almost always isolated. In complicated forms of the disease,
there is a mucopurulent exudate which contains large numbers of neutro-
phils. Focal necrosis and abscessation are frequently observed. In
septicemic pasteurellosis, the serous and mucous membranes show petechial
and ecchymotic hemorrhages, and the kidney, liver, spleen, and intestine
are congested. Pasteurella can coexist with mycoplasmal pneumonia in
swine as well as with other pathogenic bacteria in the reSpiratory
tract, such as C. pyogenes, Haemophilus, Streptococcus sp., and M.

hyorrhinis (L'Ecuyer, SWitzer, and Roberts, 1961).

Summary

Specifically, mycoplasmal pneumonia in swine and the different
names given to the disease through the years have been considered.
Steps which led to the isolation and identification of Mycoplasma
hyopneumoniae as the specific etiologic agent of this disease have
been described, as well as the organism's requirements for growth and
its distinctive morphologic features. Epidemiologic behavior of the
disease, the clinical signs and macroscopic and histologic lesions,
serologic tests such as CFT, indirect (passive) hemagglutinating
angibody (IHA), latex agglutination tube test, and immunofluorescent
antibody staining tests have been discussed. A review of the litera-
ture has defined the value of the pathologic changes observed in the
lungs of animals which died of the disease, as well as the value of
each serologic test individually as it is used to establish the diag-
nosis of mycoplasmal pneumonia in swine. Pathogenic agents other than

Mycoplasma sp. were considered for differential diagnosis.

MATERIALS AND METHODS

Lungp§pecimens

Twenty-two pig lungs were collected at a local abattoir,a of which
19 had macroscopic lesions. Eight additional pig lungs were collected
at the Animal Health Diagnostic Laboratory (AHDL), College of Veterinary
Medicine, Michigan State University, East Lansing, Michigan, from pigs
received for diagnostic purposes. Of these pigs, only 1 had macroscopic
lesions. The 30 pig lungs collected were those available when the search
for specimens was made, and no particular purpose existed regarding the
age of the pigs. In Table l are presented the pig lungs collected,
distributed by presence or absence of macroscopic lesions and the pigs'

age in weeks.

Antiserum-Conjugate-Controls

 

Antisera against M. hyopneumoniae produced in rabbits and thin lung
sections of experimentally infected pigs to be used as controls were
produced by the Animal Disease Diagnostic Laboratory, School of Veterinary
Medicine, Purdue University, West Lafayette, Indiana, using as antigen
M. suipneumoniae "J" strain ATCC No. 25934 (Armstrong, 1980). These
materials were provided through the courtesy of the above-mentioned Dr.
Armstrong, as well as fluorescein isothiocyanate (FITC) conjugate goat

antirabbit globulin, which is commercially available.b

a . . .
Milligan Packing Plant.

b . . .
Antibodies, Inc., DaVis, CA.

23

 

24

Table 1. Number of pig lungs collected and lesions

 

 

Age of Pigs With Macroscopic Without Macroscopic
(weeks) Lesions Lesions Subtotals

3 - 3a 3

5 - 1a 1

6 — 1"”1 1

8 la la 2

16 - 1a 1

20 16b - 16

24 __§_b .21” .6.
Totals 20 10 30

 

aCollected at the Animal Health Diagnostic Laboratory, Michigan
State University, East Lansing, MI.

bCollected at a local abattoir.

Normal rabbit serum was collected from rabbits at the Animal Health
Diagnostic Laboratory (AHDL) and used for negative control. Phosphate
buffered solution (PBS)C adjusted to pH 7.3, PBS-bovine serum albumin
(PBS-BSA)C used for diluting sera and conjugate, as well as buffered

. c . . '
glycerin, pH 8.5, used for mounting slides, were all prepared at the AHDL.

Macroscopic Lung Evaluation
The lungs collected at the commercial abattoir, as well as those
collected at the AHDL, were evaluated according to percentage of consoli-

dation exhibited by each of the right and left anterior lung lobe fields,

CSee Appendix, page 56.

 

 

25
and a grade was assigned according to a scheme for grading designed by

the author and here presented (Table 2).

Table 2. Grading scheme of pig lung lesions according to percent
average of consolidated right and left anteroventral

lung lobe fields

 

Percent Average of Consolidated

 

 

Anterior Lung Lobe Fields Grade
1-5 1
6-15 2
16-30 3
31-50 4
>50 5
Histopathology

 

Sections of the right and left apical lung lobes as well as the
right and left cardiac lobes were taken and fixed in buffered 10% formalin,
trimmed, embedded in paraffin, and sections 6 um thick made and stained

with hematoxylin-eosin (H&E) for histopathologic examination.

Tissues for Immunofluorescence Staining
From those lungs with macroscopic lesions, sections of consolidated
tissue were taken, and from lungs without macroscopic lesions, tissue
sections were taken at the tip of the apical and/or cardiac lobes. The
lung samples thus collected were placed in plastic bags and stored at

-20 C for periods of l to 3 weeks, until examined.

26
Indirect Immunofluorescent Staining Antibody (IFA)

From each pig lung, portions 0.4 x 0.2 cm were frozen onto sec-
tioning blocks and, with a cryostat-equipped microtome,d 2 sections 6 um
thick each, made and placed on I slide, dried for 8 to 10 minutes, fixed
in acetone for 10 minutes, and air dried.

Antiserum and normal rabbit serum, respectively, were added to
individual tissue sections to be tested, as well as to the tissue sections
used as controls. They were then incubated in a moist chamber for 30
minutes at room temperature. The slides were washed with PBS for FA
for 10 minutes, dried, and stained with fluorescein-isothiocyanate
conjugated antirabbit globulin, incubated in a moist chamber for 30
minutes at room temperature, washed with PBS for FA for 10 minutes,
and then mounting media added and coverslips applied. The slides were

. . e
examined by fluorescent microscope.

 

d . . .
International Equipment Co., Needham Heights, MA (cat. no. 31875).

e . .

Zeiss standard microscope, 100 W halogen lamp transmitted light,
iJrfrared wavelength, heat absorption filter, exciter filter, barrier
filter for FITC.

 

RESULTS

Macroscopic Lesions

The lesions described below were common to all 20 pig lungs with
variation more in degree of severity than in type of lesion. The
anteroventral parts of the apical and cardiac lobes showed macroscopic
lesions with a symmetrical tendency in distribution. Areas of consoli-
dation ranged from 5 to 60% of the lung lobe fields and appeared as
plum-colored discolored areas with a fairly firm feeling, which were
clearly demarcated from other areas of grossly normal appearing, healthy
looking lung. Consolidation was Observed extending from the tips of
the affected lobes towards the hilus. When graded for lesions, the 20
pig lungs appeared distributed as reported in Table 3. The types of

macroscopic lesions can be observed in Figures 1 through 3.

Histopathology

 

Pig Lungs with Macroscopic and Microscopic Lesions

 

The pig lungs which had macroscopic lesions showed a variety of
microscopic lesions when histologic examination was done. A summary of

morphological diagnosis is presented for each pig lung examined (Table 4).

1) Lung sections were examined from 1 pig 8 weeks old and 5 pigs
20 weeks old each. The identification numbers of these pigs in Table

4 are 1 through 6, respectively. Marked peribronchial and peribronchiolar

27

 

28

Table 3. Grading scheme of pig lung lesions according to percent average
of consolidated right and left anteroventral lung lobe fields
(number of pig lungs by grade)

 

Percent Average of Consolidated

 

Anterior Lung Lobe Fields Grade No. of Pig Lungs
1-5 1 2
6-15 2 3
16-30 3 9
31-50 4 4

>50 5 2

 

 

29

Figure l. Pneumonic pig lung. Bilateral and symmetrical consoli-
dation of the anteroventral parts of the apical and cardiac lobes.

Figure 2. Pneumonic pig lung. Consolidated right apical and
cardiac lobes.

 

 

 

30

 

Figure 2

 

31

 

Figure 3. Pneumonic pig lung. Consolidated left
apical and cardiac lobes. Lobular distribution of the
lesions. Clear-cut separation between affected lung and
normal appearing healthy lung.

32

Table 4. Summary of morphologic diagnosis and age of each study pig;
lungs with macroscopic and microscopic lesions

 

 

Subacute
Subacute to Chronic Subacute
Pig Lung Age Interstitial Interstitial Suppurative
No. (weeks) Pneumonia Pneumonia Pneumonia
l 8 X X
2 20 X X
3 20 X X
4 20 X X
S 20 X X
6 20 X X
7 20 X
8 20 X
9 20 X
10 20 X
11 20 X
12 20 X
13 20 X
14 20 X
15 20 X
16 20 X
17 20 X
18 24 X
19 24 X

20 24 X

 

33
lymphoid hyperplasia was seen in some areas, and in other areas a fairly
marked thickening of the alveolar walls with proliferation and infiltra-
tion of lymphocytes, with scarce or no indication of neutrophil infiltra-
tion was observed. Lymphocytes Were also observed in the peribronchial
and peribronchiolar spaces. Some areas of the bronchial and/or bronchiolar
epithelium had loss of cilia and desquamated epithelium, while other
areas had a hyperplastic epithelium. In other sections of lung, large
numbers of neutrophils Were observed in the alveolar spaces accompanied
by some edema. For these 6 pig lungs the microscopic characteristics
were those of a subacute interstitial pneumonia and subacute suppurative

pneumonia.

2) At histologic examination of sections of lungs of 11 pigs, 20
weeks old each, identified in Table 4 with the numbers '7 through 17,
there was an accumulation of lymphocytes, macrophages, and plasma cells
within the alveolar spaces. There was infiltration of lymphocytes and
macrophages in the alveolar walls. Loss of cilia and desquamation of
bronchial and bronchiolar epithelium were evident, and mononuclear cells
were accumulated in the peribronchial and peribronchiolar spaces.
Lymphoid hyperplasia in the bronchial tree was marked. In some lung
areas the bronchiolar epithelium was hyperplastic. Proliferating fibro—
blasts were seen in the alveolar walls. The histologic lesions described
for these 11 pig lungs were considered as a morphological expression of

a subacute to chronic interstitial pneumonia.

3) Three pigs with identification numbers l8, l9 and 20 in Table
4 had lymphocytes, macrophages and plasma cells in the alveolar lumens
on histologic examination. There Were accumulations of mononuclear cells

in the alveolar walls as Well as in the peribronchial and peribronchiolar

spaces, with prominent lymphoid hyperplasia in the bronchial tree and

 

34
proliferating fibroblasts in the alveolar walls. The characteristics
of the microscopic lesions observed and described for these 3 pig lungs

were those of a subacute to chronic interstitial pneumonia.

Pig Lungs with Only Microscopic Lesions

Ten pig lungs had no macroscopic lesions but did have microscopic
lesions at histologic examination. A summary of the morphologic diagnosis

for these 10 pig lungs is presented in Table 5.

Table 5. Summary of morphologic diagnosis and age of each study pig;
pig lungs showing only microscopic lesions

 

 

Subacute Subacute Subacute
Pig Lung Age Interstitial Nonsuppurative Suppurative

No. (weeks) Pneumonia Pneumonia Pneumonia
l 3 X

2 3 X

3 3 X

4 5 X

5 6 X X

6 8 X

7 16 X X

8 24 X X

9 24 X X

10 24 X X

 

 

35

1) Lung sections were examined from three 3-week-old pigs, one
5-week-old pig, and one 8-week-old pig, identified in Table 5 with the
numbers 1, 2, 3, 4 and 6, respectively. There were accumulations of
mononuclear cells in the peribronchiolar and perivascular spaces. The
alveolar walls appeared thickened and infiltrated by proliferations of
lymphocytes. The bronchiolar epithelium showed a moderate loss of
cilia. Collapsed lung tissue was observed in some areas of the lung
sections, and a few mononuclear cells were Observed within the alveolar
spaces. The microscopic characteristics for the above pig lung sections

examined were those of a subacute interstitial pneumonia and bronchiolitis.

2) Lung sections of 2 pigs, 6 and 16 weeks old, identified by
the numbers 5 and 7, respectively, in Table 5, were examined. There
was infiltration of mononuclear cells in the alveolar spaces with marked
thickening of the alveolar walls and infiltration by mononuclear cells
with little or no indication of neutrophil infiltration. There were
fairly marked areas of peribronchiolar and perivascular accumulations of
mononuclear cells. Loss of cilia of bronchial and bronchiolar epithelium
was observed. In other sections of the lung in both pigs, there were
accumulations of neutrophils and lymphocytes in the alveolar spaces
with some edema. In addition, there were areas showing moderate peri-
bronchiolar lymphoid hyperplasia. The lung sections of the 2 pigs showed
microscopic characteristics of a subacute interstitial pneumonia, subacute

suppurative pneumonia, and bronchiolitis.

3) In lung sections of three 6-month-old pigs examined histologically,
there were accumulations of lymphocytes within the alveolar spaces as
well as a few macrophages. These pigs are identified in Table 5 by

numbers 8 through 10. In the peribronchiolar spaces there was

 

36
accumulation of mononuclear cells (lymphocytes), as well as peri-
bronchiolar lymphoid hyperplasia. Within the alveolar walls there was
infiltration of lymphocytes and a few macrophages. The bronchial and
bronchiolar epithelium showed loss of cilia. Proliferating fibroblasts
were observed in the alveolar walls. The microscopic characteristics
in the lung sections of the 3 pigs were those of a subacute to chronic

interstitital pneumonia and subacute nonsuppurative pneumonia.

Summary_of Histopathology
In pig lungs with or without macroscopic lesions, the histologic
lesions observed were: accumulations of lymphocytes in peribronchial-

peribronchiolar perivascular spaces and alveolar lumens (Figures 4

\

through 9). Peribronchiolar lymphoid hyperplasia and discrete fibro-
blast proliferation (Figure 6), neutrophils and lymphocytes in
bronchiolar lumens (Figure 7), thickened alveoli with infiltration of
lymphocytes (Figure 8), lymphoid hyperplasia and highly dense accumula-
tions of lymphocytes in Peribronchial-peribronchiolar and perivascular

spaces (Figure 10) and loss of cilia of bronchial epithelium were seen.

Indirect Immunofluorescent (IFA) Staining Results

Discrete fluorescent particles aggregating in masses or in clusters
over the surface of the bronchial and bronchiolar epithelium were inter-
preted as M. hyopneumoniae organisms. A summary of IFA results for pig
lungs which had macroscopic and microscopic lesions is presented in
Table 6. In this category, 1 pig 8 weeks of age showed marked
fluorescence over the bronchial and bronchiolar epithelial surface.
Of sixteen 20-week-old pigs, fluorescence was observed over the surface
of the lung airways in 14, and 2 failed to show any fluorescence. Three

24-week-old pigs had fluorescence over the surface of the lung airways.

37

Figure 4. Photomicrograph of pig lung. Notice accumulations of
lymphocytes in peribronchiolar and perivascular spaces. H&E stain,
64X.

Figure 5. Photomicrograph of pig lung. Notice accumulations of
lymphocytes in peribronchiolar spaces and alveolar lumens. H&E stain,
l6OX.

38

 

Figure 4

 

Figure 5

 

39

Figure 6. Photomicrograph of pig lung. Peribronchiolar cuffing
with lymphocytes. Peribronchiolar lymphoid hyperplasia. Fibroblasts.
H&E stain, 64X.

Figure 7. Photomicrograph of pig lung. Debris, lymphocytes,
and neutrophils in bronchiolar lumen. H&E stain, 160X.

4O

 

Figure 7

41

Figure 8. Photomicrograph of pig lung. Notice infiltration of
lymphocytes in alveolar walls and bronchiolar spaces. H&E stain, 64X.

Figure 9. Photomicrograph of pig lung. Peribronchiolar accumu-
lations of lymphocytes. H&E stain, l60X.

 

42

 

Figure 9

 

43

 

Figure 10. Photomicrograph of pig lung. Notice
accumulations of lymphocytes in peribronchial-
peribronchiolar-perivascular spaces. Peribronchial-
peribronchiolar lymphoid hyperplasia. H&E stain, 64X.

44

Table 6. Summary of indirect FA test in pig lungs with macroscopic and
mitroscopic lesions and age of each study pig

 

 

 

Pig Lung No. Age (Weeks) Indirect FA Test
1 8 Positive
2 20 Positive
3 20 Positive
4 20 Positive
5 20 Negative
6 20 Positive
7 20 Positive
8 20 Positive
9 20 Positive

10 20 Positive
11 20 Positive
12 20 Positive
13 20 Positive
14 20 Positive
15 20 Positive
16 20 Negative
17 20 Positive
18 24 Positive
19 24 Positive

20 24 Positive

45
In Table 7 is presented a summary of IFA test results for pig lungs

which had no macroscopic lesions but did have microscopic lesions.

Table 7. Summary of indirect fluorescent antibody test (IFA) in pig
lungs which had no macroscopic lesions but did have micro-
scopic lesions, and age of each study pig

 

 

Pig Lung No. Age (Weeks) Indirect FA Test
1 3 Positive
2 3 Positive
3 3 Negative
4 5 Positive
5 6 Positive
6 8 Positive
7 16 Positive
8 24 Positive
9 24 Positive

10 24 Negative

 

In this category of pig lungs, in 3-week-old pigs fluorescence was
seen over the surface of the lung airways in 2 and the third failed to
show any fluorescence. Four pigs, 5, 6, 8 and 16 weeks of age, respectively,
had fluorescence over the surface of the lung airways of each one. Of
the three 24-week-old pigs, 2 had discrete fluorescence on the surface of
the lung airways while the third one failed to show any fluorescence.
The immunofluorescent test was repeated a second time for all the
pig lungs which showed only microscopic lesions. The results were as

with the first teSt, with the exception of pig 7, which was negative on

 

46
the first test and positive on the second. Those pig lungs with macro-
scopic and microsc0pic lesions which were immunofluorescent negative

were tested a second time with identical results in all cases.

DISCUSSION

Of 10 pig lungs which showed only microscopic lesions, three
3-week-old pigs showed lesions suggestive of an early M. hyopneumoniae
infection based on histopathologic findings, but fluorescence on the
surface of the lung airways was observed in only 2 of these 3 pigs,
while the third failed to show any fluorescence.

Positive results by means of the direct fluorescent staining pro-
cedure have been reported in experimental cases of the disease as early
as the 14th day postinoculation (Livingston et a1., 1972) and by the
let day postinoculation (L'Ecuyer and Boulanger, 1970). No data could
be found in the literature about diagnosis of field cases of mycoplasmal
pneumonia of swine by means of fluorescent antibody staining in very
young piglets.

As for the negative fluorescent results in pig lungs showing micro-
scopic lesions and given M. hyopneumoniae infection, reasons could be,
according to L'Ecuyer and Boulanger (1970): 1) an insufficient amount of
antigen being present to allow visualization or 2) washing out of the
antigen from the epithelium of the lung airways during the staining
procedure.

Pathogenic agents other than M. hyopneumoniae may be the cause of
microscopic lung lesions suggestive of mycoplasmal pneumonia infection
but which actually are not (Pattison, 1956; Switzer and Ross, 1975;

47

 

 

 

48

In the lungs of 2 pigs, 5 and 8 weeks old, respectively, the micro-
scopic lesions observed were suggestive of mycoplasmal pneumonia
infection, and both pigs showed fluorescence in the lung airways.

In 3 pigs 24 weeks old, the microscopic lung lesions were suggestive
of mycoplasmal pneumonia, but in addition there were microscopic changes
related to bacterial pneumonia in a stage of resolution. Two of these
pigs showed fluorescence in the lung airways, and 1 failed to show lung
fluorescence.

Of 20 pig lungs which showed macroscopic and microscopic lesions,
the lungs of l pig, age 8 weeks, and 5 pigs, age 20 weeks, showed a
variety of lesions suggestive of mycoplasmal pneumonia and superimposed
suppurative bacterial pneumonia. Fluorescence was observed in the lung
airways of 5 of these pigs; 1 pig (age 20 weeks) failed to show any
fluorescence.

In 11 pigs, age 20 weeks, and 3 pigs, age 24 weeks, the lesions
observed suggested mycoplasmal pneumonia and mild to severe superimposed
bacterial pneumonia. Of this group, 13 pigs showed fluorescence in the
lung airways and l pig (age 20 weeks) was immunofluorescent negative.

It should be pointed out that cultural procedures for isolation and
identification of pathogenic bacterial agents might have been helpful
in interpretation of the pathologic picture in the lungs. On the basis
of the studies here reported, it would appear that the indirect immuno-
fluorescent staining tests provides a rapid, relatively simple method
for the visualization of M. hyopneumoniae organisms in lung sections.

At necropsy and/or histopathologic examination of either pig lungs which
showed only microscopic lesions or pig lungs which showed macroscopic
and microscopic lesions, a suggestive diagnosis of mycoplasmal pneumonia

could be confirmed by means of indirect fluorescent antibody staining,

49
by specifically allowing the visualization of the causative agent.

For routine diagnostic purposes, isolation from tissues of M.
hyopneumoniae has been reported as difficult (Armstrong, 1980; L'Ecuyer
and Boulanger, 1970; McKean, Andrews, and Farrington, 1979) and, once
isolated, the species identification is a time-consuming process
(Armstrong, 1980; L'Ecuyer and Boulanger, 1970).

The reliability of the staining reactions obtained with pig lungs
showing microscopic lesions only or macroscopic and microscopic lesions
suggestive of M; hyopneumoniae infection would appear to be assured by:
l) the presence of fluorescence in the lung airways stained with
homologous immune globulins produced in rabbit and fluorescein iso-
thiocyanate conjugated (FITC) antirabbit globulins produced in goat,

2) the absence of such fluorescence in sections of lungs infected and
with lesions of M; hyopneumoniae when normal rabbit globulins are used,
and 3) the presence of apparently specific fluorescence in the bronchial
and bronchiolar epithelial surface in pig lungs infected with M. hyo-
pneumoniae and stained with homologous immune globulins and fluorescein
isothiocyanate conjugated antirabbit globulins produced in goats.

The FA methods have proven to be species specific (Armstrong,
1980; Kobisch, Tillon, and Vannier, 1978; L'Ecuyer and Boulanger, 1970;
Meyling, 1971).

For etiologic definitions of macroscopic and/or microscopic lesions
as caused by M. hyopneumoniae in pig lungs by means of the indirect
fluorescent antibody test (IFA), the author recommends that further

studies should be done to ascertain the degree of sensitivity of the test.

BIBLIOGRAPHY

 

 

 

 

 

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VITA

 

VITA

The author was born at La Ceiba, Honduras, C. A., on March 13,
1933. He earned his DVM degree at the University of Perugia, Perugia,
Italy, in the year 1957, and in 1974 he earned his Master's degree
in Preventive Veterinary Medicine at the University of California at
Davis, Davis, California.

Positions held by the author have included: 1) Field Veterinarian

 

(in Animal Disease Control Program), Meat Inspector, and Chief of the
Animal Health Department for the Ministry of Agriculture, Government of
Honduras, for a period of 14 years; 2) Animal Disease Control Programs
and Swine Production for the Ministry of Agriculture, Government

of Nicaragua.

The author will work in Central America.

55

 

APPENDIX

APPENDIX

Working PBS for FA

NaZHPO4 24.0 grams
NaH2P04-H20 4.4 grams
NaCl 170.0 grams
Deionized water 2000.0 ml

Each ingredient dissolved separately
Add 4 grams of NaN3

pH: 7.4

PBS-BSA (for diluting serums and conjugates)
1 gram of bovine serum albumin (BSA)a
200 m1 of working PBS
Dissolve
Pass through a 0.20 Um filter

Store at 4 C

Mountinngluid

9 parts glycerineb and 1 part PBS

 

a . .
Grand Island Biological Company, 3175 Staley Road, Grand Island,
NY 14072.

b . .
Fort Dodge Laboratories, 800 Fifth Street, NW, Fort Dodge,
IA 50501.

56

   

  
 

IIIIIIIIIII

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