BACTERIAL FLORA OF BOVINE RESPIRATORY TRACT by NICHOLAS AIGBEDO EVBUOMA

BACTERIAL FLORA OF BOVINE RESPIRATORY TRACT by NICHOLAS AIGBEDO EVBUOMA
BACTERIAL FLORA OF BOVINE RESPIRATORY TRACT
by
NICHOLAS AIGBEDO EVBUOMA
D. V. M., Ahmadu Bello University, 197b
A THESIS
submitted in partial fulfillment of the
requirements for the degree
MASTER OF SCIENCE
Department of Laboratory Medicine
KANSAS STATE UNIVERSITY
Manhattan, Kansas
1979
Approved by:
Major Prcfsssor
U
LD
TABLE OF CONTENTS
INTRODUCTION
1
LITERATURE REVIEW
2
MATERIALS AND METHOD
Collection of Specimens
9
Inoculation of Bacteriologic Media
10
Identification of Isolates
12
Statistical Analysis
13
RESULTS
l6
DISCUSSION
30
ABSTRACT
35
ACKNOWLEDGMENTS
37
LITERATURE SITED
38
APPENDICES
^
INTRODUCTION
Bovine respiratory diseases are a major source of economic loss to the
cattle industry.
It was reported that 40-80$ of all cattle diseases involve
Lillie (197*0 conservatively estimated that losses
the respiratory system.
to the Canadian cattle industry were millions of dollars annually.
It is currently accepted that a combination of factors are involved in
the etiology of the bovine respiratory disease complex (Hamdy and Trapp,
I967).
Recognized factors are stress induced by viral infection or environ-
mental factors, in combination with bacterial colonization of the lower
respiratory tract (Jericho and Langford, 1978).
Results of examination of lungs of cattle dead of the respiratory
disease complex suggest involvement of Pasteurella
Jensen et al
. ,
1976).
cultures of Pasteurella
been unsuccessful.
st>
.
(Collier, 1968, and
However, attempts to reproduce the disease with
sp_.
in animals not stressed or virus infected have
It is, therefore, difficult to assess the pathogenic role
of this group of bacteria.
Pasteurella spp
.
are recognized as part of the normal flora of the
nasopharynx and trachea of cattle (Hamdy and Trapp, 1967; Saunders and Berraan,
1964; Corstvet, 1973; and Prank and Wessman, 1978).
They have not been
recognized as a part of the normal flora of the bovine lung, but the composition of bacterial flora of the normal bovine lung has not been extensively
studied.
Collier and Rossow (1964) examined tissue from the diaphragmatic
lobe of 88 cattle at slaughter and only recovered bacteria which were
considered as inhaled soil inhabitants.
All microorganisms recovered were
considered as transient which were removed by the normal pulmonary defense
mechanisms
The purpose of this study was to examine the bacterial flora of the
normal bovine lung and to determine if recovered bacteria represented tran-
sient or resident flora.
REVIEW OF THE LITERATURE
The role of bacteria in the pathogenesis of the "bovine respiratorydisease complex was not well understood.
Most investigators suggested a
complex etiology involving a combination of bacterial, viral and environmental stress factors.
and Trapp, 196?
t
(Horlein et al.
,
1961; Collier et al .
Collier, 1968; and Gourley et al .
,
1970).
1962; Hamdy
,
Most surveys
have incriminated Pasteur ella spp . as the most predominant bacterial isolate
from pneumonic lungs (Jensen et al .
1976), although a variety of other infec-
,
tious agents including para-influenza-3 (PI-3) virus, Chlamydia sp ., adenovirus,
mycoplasmas, and infectious bovine rhinotracheitis (IBR) virus were recovered
(Collier et al.
,
1962; Horlein et al .
,
1961; and Saunders et al .
,
1964).
These species were also recovered with regularity from the nasal secretions
of diseased and healthy feedlot cattle (Horlein et al .
,
1961; Collier et al ..
1962; Hamdy and Trapp, 1967; Collier, 1968; and Gourley et al .
,
1970).
The trachea also appeared to be a residual site for these microorganisms.
Corstvet (1973) recovered Haemophilus somnus
,
Pasteurella sp.
sp . from the trachea of healthy and diseased animals.
.
and Mycoplasma
He considered these as
a part of the transient if not indigenous flora of the respiratory tract.
The presence of Pasteurella sp .
,
or other infectious agents known to be
associated with the bovine respiratory disease complex have not been
demonstrated in apparently healthy lung tissue.
Tracheal mucosa, lung hemo-
genates and bronchial lymphnodes of 88 apparently healthy cattle were examined
by Collier and Rossow (1964).
They recovered 510 isolates of bacteria and 8
isolates of common moulds prevalent in soil and feces.
Streptomyces sp . were most frequently recovered.
Bacillus sp . and
They did not isolate pasteurellae
and concluded that these were not associated with healthy tissues of the
lower respiratory tract.
None of the isolates appeared to be colonizing.
They were considered transient flora which were recently inhaled.
They
3
suggested that the lower respiratory tracts of cattle in dusty pens were
subjected to a sustained shower of soil -borne microorganisms.
Similarities
were found between organisms isolated from the respiratory tract and those
recovered from the bronchial lymphnodes.
They concluded that the lymphatic
system was important in clearance of microorganisms from the lungs.
An additional source of microorganisms in the lung was suggested by
Mullenax (1964).
He collected gas from the trachea of a cow and was able
to recover microorganisms which normally inhabited the rumen.
He concluded
that bacteria may be eructated and inhaled.
Additional studies concerning the microflora of the lung of normal
cattle were not found.
However, a number of studies were conducted on the
None or very few aerobic bacteria were
human with contradictory results.
found in most studies when specimens were obtained by bronchoscopy or
transtracheal aspiration (Pecora and Yegian, 1958, and Nozzoli and Torelli,
1975)*
In
They concluded that normal human lung parenchyma was sterile.
a more recent study, Jordan et al.
,
(1976) recovered six different genera
of aerobes and seven different genera of anaerobes when specimens of tracheo-
bronchial secretions were collected by fiber-optic bronchoscopy.
Lindsay
and Pierce (1978) examined the hypothesis that normal lung was sterile.
They utilized the dog as a model and recovered aerobic bacteria from J7%
of 268 lung samples from 19 dogs.
They postulated that the lung was not a
flawlessly sterile environment because bacteria from the pharynx were
continuously aspirated, especially during sleep.
Some of these bacteria
were neither killed nor eliminated immediately by host defenses.
They
replicated in normal lung where they remained for varying intervals.
It
was not clear whether establishment of organisms in the lung was due to
aspiration of unusually large numbers of organisms, a defect in the host
defenses or both.
They did not find evidence to support the theory that
more bacteria occured in the ventral portion of the lung.
The pulmonary defense mechanisms were considered adequate to ensure
sterility of the normal lung (Kaltreider, 1976).
It was only when these
mechanisms were impaired that microorganisms colonized and proliferated
(Green, 1968).
It was postulated that the dynamics of deposition of inhaled particles
in the respiratory tract obeyed the physical laws of inertia (Gareth and
Green, I967).
This implied that the smaller the particle, the more
distal it was deposited.
It was calculated that
with a diameter greater than
"fysn
90fo
of inhaled particles
were deposited on the mucosa from the
distal bronchiole to the nasopharynx while 90$ of those between 0.5 to
3um were deposited in the alveoli and respiratory bronchioles.
Particles
of less than 0.5um were usually not deposited and remained suspended in
exhaled. air (Kaltreider, 1976).
Jericho and O'Connel (197*0 studied the deposition of Bacillus subltlis
var. niger spores in the respiratory tract of cattle
and nasal instillation.
W
following inhalation
Inhaled aerosolyzed spores were deposited more in
the posterior segments of the lungs than spores in liquid suspension which
were instilled intranasally .
Statistical analysis of his results indicated
that inhaled aerosolyzed spores were equally deposited in any segment of
the tract.
This seemed to contradict the findings of Lillie and Thompson
(1972) who exposed calves to aerosols of P. hemolytica .
They found fewer
microorganisms in the posterior portions of the diaphragmatic lobe than in
other parts of the lung.
The mechanism of pulmonary clearance of inhaled particles has been
thoroughly studied.
Appreciation of this mechanism would be relevant to
understanding the respiratory tract environment.
A filtering mechanism
which served to trap large particulate matter suspended in inhaled air
was present in the nasopharynx of mammals (Sisson and Grossman (ed) i960).
One to seven per cent of aerosolyzed P. multoclda were recovered from
bovine lung tissue homogenates when administered by inhalation whereas
40-80# were recovered after intra-bronchial injection (Flossman, 1977).
The respiratory tract mucosa was coated by a mucus secretion that was
of a special physical consistency.
It contained proteolytic enzymes and
offered physical, chemical, and immunologic barriers to invading microorganisms
(Kaltreider, 1976).
It flowed anteriorly, moved by the biphasic whiplike
motion of the cilia of the epithelial cells that lined the respiratory
tract from the distal bronchiole to the nasopharynx (Green, 1968).
It was
described as an escalator because It carried deposited particles from the
distal bronchioles to the nasopharynx (Green, I968).
man was measured at 10-20 mm/rain.
,
The rate of movement in
culminating in clearance of more than
90% of total deposited material in less than 60 minutes (Kaltreider, 1976).
Lillie and Thompson (1972) compared the rate of clearance of bacteria
from the lungs of white mice and calves.
these agents more rapidly than mice.
mucociliary activities.
They found that calves cleared
The difference was not attributed to
Pulmonary macrophages were concluded to be more
active in bovine than in murine lungs.
Non ciliated epithelial cells lined the mucosal surfaces of the respiratory bronchioles and alveoli of mammals (Kaltreider, 1976).
Inhaled
particles deposited in these regions were removed by more complex systems.
The rate of fluid flow in these regions was very slow and rated in days
and years (Kaltreider, 1976).
stood.
The mechanisms of flow were poorly under-
Alveolar macrophages played a dominant role in removal of particu-
late matter from these regions.
Those which were ladden with engulfed
particles migrated to the distal bronchioles from where they were carried
to the nasopharynx via the mucociliary escalatory mechanism.
Those
particles not engulfed by alveolar macrophages were drawn into the lymphatic
drainage system at special areas on the mucosa described as "lympho-
epithelial organs".
These blind pocket origins of lymphatic ducts exerted
negative pressure on the content of the alveoli and respiratory bronchioles
(Kaltreider, 1976).
Such particles, if they persisted and were not degraded,
usually ended up in regional lymph nodes.
Particles not removed by either
of the above mechanisms penetrated the respiratory epithelium and entered
the interepithelial connective tissue where they were engulfed by histio-
cytes (Kaltreider, 1976).
Living particulate matter such as bacteria and viruses, were rapidly
neutralized in a specific manner by the immune defense mechanism of the
respiratory system.
This system has been extensively studied.
Immuno-
globulins of the IgA, lgG, lgM and lgE classes were reported present in
respiratory tract secretions of the dog (Kaltreider, 1976).
These anti-
bodies occured in a relatively higher concentration in pulmonary secretions
than could be explained by transudation from intravascular fluid.
Immuno-
fluorescence studies of submucosal lymphoid tissue indicated local production (Martinez. - Tello et al., 1968).
Immunoglobulin G was most effective in combating bacterial invasion of
the lower respiratory tract.
It fixed complement which was demonstrated to
be present in low levels in normal bronchial secretion but increased with
inflammation (Johnson and Philip, 1977).
Alveolar macrophages had receptor
sites for the Fc portion of IgG molecule which facilitated bacterial
opsonization (Fundenberg et al.
,
(ed) 1976).
Immunoglobulin A was most effective as a neutralizing antibody but
less effective in combating bacteria.
It blocked receptor sites on invading
microorganisms, thereby preventing them from attaching to mucosal surfaces.
It neutralized inhaled toxic macromolecules and exerted an antibacterial
effect in conjunction with lysozyme or lactoferrin (De Coteau, 197*0.
Cell mediated immunity was involved in the defense mechanism of the lung.
Locally produced T-cells elaborated lymphokines that affected alveolar
7
macrophages (Johnson and Philp, 1977).
The latter became activated and more
competent in their ability to destroy bacteria.
Gerbrandy and Dura (1972) demonstrated an anamnestic response involving
immunoglobulins in the respiratory tract.
Gadol and Johnson (1974) con-
cluded that pulmonary T-lymphocytes exhibited memory, but pulmonary B-
lymphocytes did not.
Certain agents were known to impair pulmonary defense mechanisms.
Para-influenza-3 (PI-3) virus destroyed cilia lining the upper respiratory
tract.
It was also shown to impair ingestion and killing of bacteria by
mouse alveolar macrophages (Warshaur, 1977).
In the latter case, the
reaction was optimum when mice were challenged with bacteria 7 to 11 days
post exposure.
There was no noticeable impairment of macrophage activity
when mice were challenged on post-exposure day three.
Inert dust did not enhance bacterial colonization in hamsters, although,
it seemed to favor infection of the lungs by mycoplasma (Battigel, 1971).
Ozone, as a pollutant, decreased pulmonary bactericidal effects (Gold-
stein et aL.
,
1971).
In humans tobacco smoke caused production of large
amounts of activated macrophages (Johnson and Philp, 1977).
enzymes caused damage to pulmonary tissues when released.
retarded alveolar macrophage activities (Marc-Laf orce
,
Their lysosomal
Pulmonary edema
1973).
The pathogenesis of shipping fever pneumonia in cattle has not been
well understood.
In humans, presence of low levels of bacteria in the lungs
was reported as a possible cause of emphysema (Lindsay and Pierce, 1978).
Some bacteria were reported to have alpha-anti trypsin inhibitory capacity
in vitro.
Others produced a mild secondary inflammatory response which
incited release of macrophage or leukocyte protease (Lindsay and Pierce,
1978).
Tissue destruction resulting from subsequent enzymatic degradation
of lung tissue might lead to the development of emphysema.
8
Jensen (1976) hypothesized that endotoxin from Pasteurella sv
.
formed
thrombi which occluded lymphatics, capillaries and veins in infected lobules
resulting in ischemic neorosis.
MATERIALS AND METHODS
Collection of Specimens
Fluids were collected on sterile cotton tipped applicators from the
tracheal and bronchial mucosa of 50 "bovine lungs at slaughter.*
The beef
cattle from which specimens were collected were of assorted sex and breed.
The animals were estimated at
18-2*J-
months of age and graded good to prime.
Animals were slaughtered at a rate of approximately 300 per hour, and
continually arrived at the plant by truck.
No attempt was made to select
animals from a particular area and they originated from several feedlots
in Kansas and Nebraska (figure 2).
Two or four lungs were collected and examined at one or two week
intervals from July to November, 1978.
Specimens were collected only from
lungs which were free of gross lesions and animals on which edible parts
were passed for human consumption.**
Spillage of gastro intestinal content
onto any part of the viscera was selected as a criterion for rejection of
the lung.
Estimated time from stunning to evisceration was 30 minutes.
Immediately after evisceration, the selected lungs were removed from the
line, taken to a clean area and placed in a sanitized plastic container.
The trachea and bronchi were opened with sanitized scissors which were
placed in
9%
sampled.
Fluids were collected from the mucosa of ten portions of the
ethanol and flamed before cutting into each area of the lung
tracheo -bronchial tree (figure l).
Areas sampled weret
(a)
The trachea at a level 12-13 cm cranial to its bifurcation
(b)
The tracheal bifurcation
*Iowa Beef Processors, Inc., Emporia, Kansas.
**United States Department of Agriculture, Meat Inspection Division
10
(c)
The distal bronchi at a level just large enough for passage of
the applicator (approximately 4 mm diameter) in the following
regionst
(1)
Right cranial apical lobe
(2)
Right caudal apical lobe
(3)
Cardiac lobe
(4)
Right diaphragmatic lobe
(5)
Accessory lobe
(6)
Left cranial apical lobe
(?)
Left caudal apical lobe
(8)
Left diaphragmatic lobe
Following collection, swabs were immediately placed into 1 ml of sterile
phosphate buffered saline (PBS) in screw capped tubes.
The portion of the
swab in contact with the hand was broken off and discarded.
Collection of
specimens from four lungs required approximately 90 minutes.
Inoculation of Bacterlologlc Media
Within 20 minutes of collection of the last specimen, they were taken
to a local laboratory* for culturing.
Each tube was agitated on a vortex
mixer for 30 seconds to suspend fluids and bacteria in the PBS.
The swab
was pressed against the side of the tube to express excess PBS, aseptically
removed and transferred to k ml of Tryptic soy broth**.
Four drops of the
PBS were then placed on the surface of four or five different culture
media in plastic disposable petri dishes,*** and streaked for isolation.
Department of Bacteriology, Emporia State University, Emporia, Kansas.
**Difco Laboratories, Detroit, Michigan.
***Fisher Scientific Co., St. Louis, Missouri.
11
The primary plating media utilised were»
(1)
Blood Agar (BA) - Trypticase soy agar* plus
%
citrated
bovine "blood.
(2)
MacConkey Agar** (MAC)
(3)
Phenylethyl Alcohol Agar (PEA)* plus
%
citrated bovine
blood
(k)
Chocolate Agar (CA) - Trypticase soy agar* plus 1% Hemoglobin**
and 1% IsoVitalex**
OR
Lysed Blood Agar
(LBA.)
Trypticase soy Agar* plus 10#
-
citrated bovine blood which had been frozen and 0.25 gm per
litre BETA DPN***.
Thayer-Martin Agar (TM)
(5)
-
Mueller-Hinton Agar** plus 1% Hemo-
globin**, 1% IsoVitalex** and 1% V-C-N Inhibitor* (Vancomycin
300 mg. Colistin 750 meg and Nystatin 1,250 units per ml).
Following sampling of the tenth lung, the supply of IsoVitalex** was
exhausted.
Attempts to replenish the supply were unsuccessful at that time
because all local suppliers were unable to obtain the product from the
manufacturer**.
At that point, lysed blood agar was substituted for
chocolate agar and Thayer-Martin agar was dropped as a primary plating
medium.
All batches of both chocolate agar and lysed blood agar were
tested for their ability to support the growth of an "X" and "V" factor
requiring Haemophilus sp
.
and Haemophilus somnus throughout the project.
The petri dishes were packed into polyethylene bags and stacked horizontally
in an empty ice chest for the trip back to Manhattan, Kansas.
Baltimore Biological Company, Baltimore, Maryland.
**Difco Laboratories, Detroit, Michigan.
***Sigma Chemical Company, Baltimore, Maryland.
12
Within two hours of the time of plating the last specimen, the plates
and tubes of TSB were placed in a 37C aerobic incubator with a
Co- tension.
%
increased
Tubes of TSB were incubated for 24 hours and one loopful
streaked for isolation on Mannitol Salt Agar* plates and incubated at 3?G.
Identification of Isolates
Following 18, 48 and 72 hours incubation, plates were examined and
each colony type enumerated, described and subcultured.
.
Each different
type of microorganism recovered was preserved by freezing on glass beads
at -60C (Nagel and Kunz, 1972).
Pure cultures of each microorganism were
identified when possible, using generally accepted procedures and keys
(Buchanan and Gibbons (ed.) 1974; Gordon et al .
1974; Kloos et al .
,
,
(l973)l Kloos and Schleifer,
1975? Lennet et al., (ed.) 1974? Smith and Bettge (1972);
Schleifer and Kloos, 1975; Weaver et al
*Dlfco Laboratories, Detroit, Michigan.
.
,
(1974).
13
Statistical Analysis
The enumerated colony forming units from one petri dish of a particular
medium on which the microorganism grew, was most numerous and well isolated,
were utilized for statistical analysis.
These numbers were analyzed in an attempt to determine whether microorganisms recovered from various locations in the lung were inhaled and
transient residents or were actively colonizing and proliferating in the
fluids.
This analysis was based on the assumption that a definite number
of microorganisms of a particular type should be recovered from a particular
location to be considered as colonizing.
It was assumed that in the absence
of colonization, the distribution of an organism in respect to numbers
recovered would be random and that this rendomness would fit a generalized
Poisson probability model (Cohen, I960).
The procedure adopted for fitting
a Poisson probability model to the data was sequential.
The model was
first fitted to all data collected on each genus of bacteria collected
at a sampling site.
A chi square
goodness of fit (Snedecor and Cochran (ed)
I967) was used to test the adequacy of the model.
was truncated by removal of the most extreme class.
When inadequate, the data
Truncation continued
until an ordered subset of classes starting at X=0 to X= an indefinite
number (k) was found which adequately fit the Poisson model.
Where X
stands for the number of colonies of a particular genus isolated from a
particular sampling site.
The probability of making each observation of
X under the adopted model was calculated.
For example of the method, see
appendix (table 51 )•
To determine if any particular area of the tract was more predominantly
colonized, a chi square goodness of fit for uniform distribution was
utilized (Snedecor and Cochran (Ed.) I967).
FIGUBE I
Diagrammatic representation of the bovine respiratory
tract (Dorsal view).
Locations from which secretions
were collected are designated A-J.
A - Trachea
F - Right diaphragmatic
B - Tracheal "bifurcation
G - Accessory
C - Right cranial apical
H - Left cranial apical
D - Right caudal apical
I - Left caudal apical
E - Cardiac
J - Left diaphragmatic
14
Figure
1
FIGURE
2
Distribution of feedlots on which examined
cattle were raised.
15
Figure 2
C/5
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c
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LLJ
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+*
—
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•
r
•
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o
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16
RESULTS
Aerobic bacteriological examination of tracheal and bronchial fluids
from 50 bovine lungs resulted in recovery of bacteria belonging to 20
genera.
Additional isolates were placed into two Centre for Disease Con-
trol (CDC) alpha-numeric designations.
Microorganism recovery frequencies
by location sampled are presented in Table 1.
A total of 433 isolates was recovered from 48 of the respiratory tracts.
Two of the tracts failed to yield growth of any bacteria from any of the
locations sampled.
More isolates were recovered from the trachea than any
other single location.
They accounted for 112 (25. 9#) of the total isolates.
The second most frequent source of Isolates was the tracheal bifurcation
80 (18. 5#).
Other locations sampled yielded from 22 (5.1%) to 38 (8.890
of the isolates (Table 4).
Based on the statistical analysis, a total of 146 of the recovered
isolates were considered as colonizing the tracts (Table 2)
.
The number
of organisms isolated from a given location which were considered as colonizing varied markedly from one bacterial genus to another and from one
sampling site to another.
This number varied from as few as three colony
forming units (CFU) to as many as too numerous to count.
Eight of the tracts did not yield enough CFU's of any one microorganism in any single location to be considered colonized.
Since there was
no growth from two tracts, forty tracts were considered colonized.
Twenty
of the tracts were colonized by only 1 genus, 5 by 2, 10 by 3, 3 by 4, 1 by
5 and 1 by 9.
Seventeen genera and one CDC alpha-numeric designation recovered were
considered colonizing.
ted in Table 2.
The frequency of colonization by location is presen-
Members of the genus Streptomyces were found most frequently
-
.
1?
as a colonizer (29. 5#).
isolates.
Pasteur ella
st> .
represented 13$ of the colonizing
These species colonized a total of 19 locations in 9 tracts.
The sites most frequently colonized by Pasteurella
sp_.
were the trachea and its
bifurcation, but they were recovered in at least one instance from all but
two (left cranial apical and left diaphragmatic) sites.
Pasteurella so .
were the second most widely distributed colonizing microorganism recovered.
They were recovered from an additional 9 tracts, but not in sufficient
numbers to be considered colonizing.
The frequency of colonization of the various sampling sites without
The trachea and tracheal bifurcation
regard to genus is presented in Table 3«
They were colonized more frequently
were the areas most frequently colonized.
than other locations at a probability (P) less than 0.001 by the chi square
test for goodness of fit for uniform distribution.
They were not signifi-
The frequency of colonization of other
cantly different from each other.
sampling sites ranged from 4 to 11 of the 50 lungs sampled.
There was no
significant difference (P=,63) between the degree of colonization in these
sites
GDC Alpha-numeric Designations t
by lie.
Groups
Hf
Organisms in this class were recovered
One lung was colonized in the right caudal apical lobe
at low frequencies.
,
lib and IVf were recovered from a total of k tracts
and were considered transient flora.
Gram Negative Aerobic Rods
:
A single colony of Bordetella bronchi
se-ptica was recovered from the accessory lobe.
were recovered from a total of 9 lungs.
Six species of pseudomonads
One lung was colonized in the left
diaphragmatic lobe by Pseudomonas acidovorans .
The pseudomonads comprised
2.3$ of total isolates.
Gram Negative Cocci and Goccobacilll
represented 7.3$ of total isolates.
only as Neisseria
sp_.
:
Members of the genus Neisseria
Twenty-eight isolates were identifiable
One particular isolate was very dysgonic.
It was
18
wet, flat and irregular.
At 18 hours incubation, colonies were 5mm in
diameter with a narrow zone of complete hemolysis.
They did not survive preserva-
zing the trachea and tracheal bifurcation.
tion and were not further characterized.
They were found coloni-
Neisseria mucosa was recovered
from the left apical lobe, and Neisseria sicca colonized the right diaphragmatic.
Branhamella cataxrhalls was recovered from 2 lungs .
It was found
along with P. hemolytica colonizing the trachea, tracheal bifurcation and
accessory lobe of one lung.
Eight isolates of moraxellae were identified to the species level.
Pour isolates were referred to as Moraxella
speciated using available keys.
sp_.
because they could not be
Seven tracts were colonized in 8 locations
by members of the genus Moraxella .
Gram Negative Facultatively Anaerobic Rods i
of pasteurellae were recovered.
Three recognizable species
Pasteur ella hemolytica represented 61.8%
and P. Multocida 32.4$ of total pasteurellae isolates.
nanlum was recovered once from the trachea.
Pasteur ella sv .
Pasteur ella galli-
One isolate was identified as
It produced
It was a Gram-negative pleomorphic bacillus.
acid over acid on triple sugar iron agar* (TSl), a positive oxidase reaction
and reduced nitrate.
It was indole, urea and citrate negative.
produced in 1% glucose and maltose in heart infusion broth*.
Acid was
It did not
produce acid in 1% xylose, mannitol, lactose or sucrose.
Enterobacteria were recovered at low frequencies.
Enterobacter llque-
faciens colonized two lungs of cattle from the same feedyard.
Escherichia
coll was isolated from three lungs, but colonized the trachea of only one.
Three isolates of a Haemophilus-like organism colonized the trachea and
tracheal bifurcation of one lung and the tracheal bifurcation and left
*DIFCO Laboratories, Detroit, Michigan
.
19
diaphragmatic lobe of another.
gram-negative small rods.
These organisms were very dysgonic and were
A twenty -four hour growth on
"blood agar
produced
colonies that were round, greyish, glistening, smooth, slightly raised and
A complete zone of hemolysis surrounded each colony.
1-2 mm in diameter.
The cultures did not grow on any differential
They were oxidase positive.
media without the addition of serum.
They produced indole and produced
acid from maltose, xylose, lactose, sucrose, mannitol and glucose.
Aeromonas hydrophila colonized the diaphragmatic lobe of one lung.
Gram Positive Cocci
;
Ten per cent of the isolates were staphylococci
Goagulase positive staphylocci were not recovered.
All recovered staphy-
lococci were enumerated on any of three primary plating media, namely BA,
PEA and, LBA.
There was no occasion when staphylococci were isolated on
mannitol salt agar without being isolated at the same time on any of the
previously mentioned media.
All
*+?
isolates were identified as to species.
Staphylococcus epidermidis was the most predominant.
Five other species
were isolated with less frequency.
Micrococcus sp
.
comprised 6% of all isolates.
They were recovered from
20 lungs and were separated from the staphylococci by their inability to
produce acid aerobically from 10$ glycerol in purple agar base plus ^mg/
litre of streptomycin (Schleifer and Kloos).
Five serological groups of streptococci were recovered.
9% of total isolates.
scheme.
These comprised
They were classified according to the Lancefield
Group D was the most predominant.
A single colony of Group A
streptococcus was recovered from the trachea of one animal.
Sndospore forming Rods:
Eight species of Bacillus were identified.
Bacillus pumilus and B. subtillis predominated and were recovered from 12
and 11 lungs respectively.
Members of the genus Bacillus comprised 10^
of total isolates, but colonized only 3 lungs.
20
Gram Positive Asporogenous Rod-shaped Bacteria:
Lactobacilli were
recovered from the tracheal bifurcation on two occasions.
However, conditions
of this study were not optimal for recovery of these organisms.
Actinomycetes and Related Organisms :
class of organism recovered.
This was the most predominant
Corvnebacterium
sp,.
comprised 11^ of all isolates,
and were recovered from 31 lungs, and colonized 10.
No recognizable species
was identified and were best classified as diphtheroids.
Streptomyces sp . were the most numerous genus and represented 23^ of
all isolates.
They were recovered from 30 lungs and colonized 15.
the lungs were colonized in all examined locations.
speciate members of this genus.
Two of
No attempt was made to
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Table 3. Frequency of colonization of different locations in
the respiratory tract of 50 cattle.
Location
Number of Lungs
Colonized
Percentage
Trachea
25
50%
Tracheal bifurcation
2^
±8%
Right cranial apical lobe
4
Q%
Right caudal apical lobe
6
12#
6
12*
Cardiac lobe
9
^%
lW
Left cranial apical lobe
5
10 #
Left caudal apical lobe
7
^%
Left diaphragmatic lobe
11
22%
Right diaphragmatic lobe
Accessory lobe
9
29
Table *+. Isolation frequency of aerobic bacteria from various
locations in the respiratory tracts of 50 cattle.
Location
Number of Isolates
Percent of Total
Isolates
112
25.9
Tracheal bifurcation
80
18.5
Right cranial apical lobe
28
6.5
Right caudal apical lobe
28
6.5
22
5.1
36
8.3
31
7.2
Left cranial apical lobe
33
7.6
Left caudal apical lobe
25
5.8
Left diaphragmatic lobe
_J8
8.8
Total
if33
Trachea
Cardiac lobe
Right diaphragmatic lobe
Accessory lobe
30
DISCUSSION
Four hundred thirty-three Isolates of bacteria, some of which were known
pathogens were recovered from fifty bovine lungs in varying numbers and
frequencies.
Thus a hypothesis that the lung is sterile (Pecora and Yegan,
1958) cannot be advanced for feedlot cattle.
The ecological status of re-
covered bacteria varied from transient to colonization.
It was apparent
that the pulmonary defense mechanism was not adequate to maintain sterility
within the respiratory tract.
studied.
The reasons for this would need to be further
A few possibilities will be discussed.
The concentration of microorganisms in inhaled air especially in dusty
pens could overwhelm the mechanisms of pulmonary clearance.
with the suggestion of Collier and Bossow, (196^)
,
This conforms
who examined the respi-
ratory tracts of 88 healthy cattle at slaughter and recovered 510 isolates
of bacteria prevalent in soil and feces.
Cattle raised under feedlot conditions may be defective in pulmonary
defense mechanism.
If the rate of clearance of particulate matter by the
mucociliary escalatory mechanism in the bovine is comparable to that estab-
lished for man, no bacteria would be able to stay long enough in the
respiratory tract to proliferate and colonize.
However, if there was a flaw
in the mechanism, the clearance of inhaled particles would not be thorough.
Such a flaw could be due to presence of areas on the respiratory tract
mucosa lined by epithelial cells that lack cilia.
Such places could serve
as "islands" on which microorganisms might have settled and proliferated.
The "lymphoepithelia organs" (Kaltreider, 1976) could serve as such a site.
Similar organs in the gastro-intestinal tract of mouse (lymphoepithelium overlying
Peyer's patches) have been recognized as sites for bacterial proliferation
(Abrams, 1977).
Viruses have been reported to destroy ciliary epithelium
;
31
lining the upper respiratory tract (Jericho and Langford, 1978).
Tracheo-bronchial secretions in feedlot cattle may be exceptionally rich
in substances which could be utilized by some bacteria for growth.
This
speculation was raised because some organisms that were dysgonic on enriched
laboratory media were recovered from the respiratory tract in numbers that
indicated they were growing luxuriantly in fluids of the tract.
The immune systems associated with the respiratory tract of beef cattle
may be defective.
Some organisms that produce mucinase could penetrate the
physical barriers offered by the mucus lining of the respiratory tract,
attach to the surface of epithelial cells and proliferate.
Vibrio cholera
and Campylobacter fetus are known to have mucinase activities (Burnet
Dennis, 1967).
,
19^8
The trachea and tracheal bifurcation which were more fre-
quently colonized than the rest of the tract appeared to be the source of
organisms that later colonized the lower regions.
As there was no signifi-
cant difference in levels of organisms recovered from the different lobes
of the lung, a rational conclusion would be to postulate that organisms
were carried to these regions suspended in inhaled air.
Particles that are
thus distributed have equal chances of being deposited within any lobe.
If
they were distributed as a suspension in aspirated fluid, they would tend
to concentrate in a ventrally situated lobe such as the accessory lobe.
On a few occassions, organisms that were not recovered from the trachea
were found colonizing the bronchi.
An example was Aeromonas hvdrophila
which was recovered from the left diaphragmatic lobe in numbers that were
too numerous to count and was not recovered from any other location.
In
such an instance, it was possible that the organism invaded the lung via a
hematogenous route or it may have been present in inhaled air in such low
numbers that only one organism was deposited at a site which it colonized.
On the other hand, this isolate could have been deposited in the trachea,
32
but did not survive due to unfavorable conditions, one of which might be
competition with numerous other organisms for growth factors.
An important result obtained from these studies was the recovery of
Nine of the lungs were
from 18 of the 50 lung3 examined.
Pasteurella
sp-p
colonized.
Collier and Rossow (1963) reported that Pasteurella svv . were
.
The re-
not associated with apparently healthy lung tissue in the bovine.
sults of this study contradicted the above.
Pasteurella
st>p .
were found
It is
colonizing at least once in eight of the ten locations examined.
possible that the stress to which animals were subjected prior to slaughter
may have contributed to colonization of the bronchi by pasteurellae.
Gavallero and Sala (1951) described the effect of corticosterone on latent
pasteurella infection in mice.
This substance is released in the body as
a result of physiological stress and has been implicated as a possible
cause for enhancement of pasteurella infection.
It was reported that some strains of pasteurellae were virulent while
others were not (Carter and Bain, I960)
in this survey were not serotyped.
.
The pasteurellae that were found
There were no gross pathologic lesions
associated with the lungs which were colonized.
these isolates was not determined.
The potential virulence of
However, virulence factors which have
been extensively studied in some species of bacteria are not well known in
pasteurellae.
Plasmids are known to encode for the synthesis of various
factors in Escherichia coli (Magdalene et al .
Bouanchaud et al
. ,
1975:
,
1978;
Orskov and Orskov, 1973)
•
Gyles et al
.
,
1978?
It is known that such
plasmids are transferable during conjugation or by lysogenic bacteriophage.
Takeda and Murphy (1978) demonstrated the conversion of a non-enterotoxigenic 2. coll to an enterotoxigenic strain by the latter method.
Bacter-
iophage have been associated with pasteurella (Gadberry and Miller, 1978).
Similar studies, if applied to Pasteurella could yield valuable results.
.
33
Physiological stress was believed to increase susceptibility of animals
to pasteurellae infection and heighten virulence of the organism (Carter and
Bain, I960).
Cavalero and Sala (1951) demonstrated the effect of steroid
hormones on the virulence of P. multoclda in rats.
Latent infections were
made acute and lethal by inoculation of cortisone, a substance which is re-
leased in the body as a result of physiological stress.
This suggests that
physiological conditions may alter the characteristics of pasteurellae.
A
saprophytic phase could turn parasitic and virulent if provided with favorable conditions.
It would thus be considered an opportunistic pathogen.
The mechanism by which Pasteur ella svv . cause diseases is not well
known.
A toxic pyrogenic lipopolysaccharide was Isolated from type B strain
of P. multocida (Carter and Bain).
Jensen et al .
,
1976, suggested that
pasteurellae endotoxin formed thrombi which occluded lymphatics, cappillaries
and veins resulting in ischemic necrosis of the infected tissues.
Hemophilus somnus was not recovered from the respiratory tract in this
study.
Corstvet et al .
,
(1973) found H. somnus in the trachea of living
feedlot cattle and concluded that they were part of the transient, if not
indigenous, flora of the respiratory tract.
Two organisms were recovered which were classified as Haemophiliuslike.
They grew in heart infusion broth to which serum was added.
possible to determine that they formed acid from maltose.
It was
Recent reports
indicated the possibility of false positive maltose reactions in serum-
supplemented media.
This was attributed to the presence in serum of a sub*
stance which hydrolysed maltose to two molecules of glucose.
*Hollis D.G., Riley P.S., and R.E. Weaver. Center for Disease Control,
Atlanta, Georgia. Serum supplementation as a cause of false positive
maltose reactions
Amended Description of Kingella denitrifleans
Abst., 79th Annual Meeting, American Society for Microbiology, Los Angeles,
California, 1979.
«
3^
Streptomyces were the most frequently recovered organisms.
Their
presence in the respiratory tract would suggest inhalation of soil-borne
particles since this group of microorganisms is commonly found in the soil
(Stanier et
al,. ).
Some species of streptomyces produce potent antibacterial
substances (Stanier et al. ) that could possibly preclude establishment of
other bacteria within the niche.
The phenomenon of bacterial interference
as it affects the respiratory tract of feedlot cattle should be further
studied.
There is not sufficient evidence in this survey to conclude that
the presence of one organism excludes another from the same location.
The numbers of a particular organism that were recovered from a location and statistically determined as colonizing varied from one bacterial
genus to another and from one sampling site to another.
This was to be
expected on the basis of the prevalence of the bacteria in the environment
and the non-independence between sampling sites.
It was not possible to identify all organisms as to species.
Coloni-
zation was therefore determined on the basis of genera of organism.
Statistical analysis to determine colonization assumed a Poisson probability
model for the randomness of distribution of an organism in a sampling site.
This analysis may be in error to the degree that this model was fitted to
the distribution of a genus of organism instead of the species.
BACTERIAL FLORA OF BOVINE RESPIRATORY TRACT
by
NICHOLAS AIGBEDO EVBUOMA
D. V. M., Ahmadu Bello University, 1974
An abstract of
A THESIS
submitted in partial fulfillment of the
requirements for the degree
MASTER OF SCIENCE
Department of Laboratory Medicine
KANSAS STATE UNIVERSITY
Manhattan, Kansas
1979
.
35
ABSTRACT
Bovine respiratory disease is a cause of great economic loss in the
cattle industry.
Its etiology has been attributed to a combination of complex
factors which include viruses, bacteria and environmental stress.
sp .
Pasteurella
are the most frequently recovered bacterial isolate, but its association
with apparently healthy cattle lung tissue has not been reported.
The respiratory tract of 50 cattle were examined at slaughter for
aerobic bacteria.
Cotton tipped applicators were used to obtain tracheal and
bronchial fluids from ten locations in each lung.
Two locations in the trachea
and eight locations corresponding with the distal bronchi of the major pulmonary lobes were examined.
Four or five selective and differential media
were used for pirmary isolation.
A total of ^33 isolates comprising 22 genera were recovered.
myces
sp_.
Strepto -
were the most frequently recovered and represented 22. 7# of the
total isolates.
Pasteurella sp . represented 7.8^ of all isolates and were
recovered from 18 lungs.
They were considered transient in nine and coloni-
zing in nine.
The trachea and tracheal bifurcation were most frequently colonized by
bacteria.
There was no significant difference between levels of colonization
of different lobes of the lungs.
Pasteurella sp . colonized the trachea and
tracheal bifurcation more frequently than the bronchi.
It was hypothesized that
physical stress to which animals were subjected prior to slaughter may be
responsible for colonization of bronchi by pasteurellae
It was concluded that the respiratory tract of apparently healthy beef
cattle was not a sterile environment.
The region from the trachea to the
distal bronchi were colonized by various species of bacteria, most of which
had their origin in the soil, feces or pharynx.
These organisms persisted
in spite of the elaborate mechanism by which the lung rids itself of particulate
matter.
This mechanism was not adequate to maintain sterility.
It was not
certain whether this inadequacy was due to an inherent defect that was peculiar
organisms
to beef cattle or due to the presence of an overwhelming number of
in inhaled air.
It is recommended that the pulmonary defense mechanisms in
feedlot cattle be further studied.
37
ACKNOWLEDGMENTS
professor, Dr. W. E.
I wish to express my sincere gratitude to my major
and construcBaillie for his help in obtaining specimens, valuable guidance
tive criticisms.
Appreciation is also extended to Dr. E. H. Coles, Labora-
Laboratory, as
tory Animal Medicine, and Dr. H. D. Anthony, Diagnostic
members of committee and for reviewing the manuscript.
of Laboratory
I am grateful to the entire staff of the department
Animal Medicine for bearing with me during the course of this study.
My
Laboratory
special thanks go to Mr. E. C. Stowe, Med. Tech. Department of
B.
Animal Medicine, for his invaluable technical assistance, Dr. Lynette
Corbeil for providing research papers, and Dr. Robert Corbeil for helping
with statistical analysis of results.
Research
Finally, I wish to thank the National Institute for Veterinary
in Nigeria for providing the funds for my studies and the Kansas State
University for providing research facilities.
38
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44
APPENDICES
45
CALCULATION USED TO DETERMINE COLONIZATION
As an illustrative example of the procedure used in determining
colonization, suppose that in the tracheal bifurcation, the following
number of corynebacterium Isolates were observed in 50 respiratory
tracts.
0123^56789
532010001
Xi*
Observed frequency f*=
37
Relative frequency mix
.71*
.04
.10 .06
.02
.02
10
1
.02
This particular sample had a mean x=0.82 isolates per respiratory tract
The generalized Polsson model is (Cohen, 1960)1
at the given site.
e
Pr (X o *0) = e' (l-«x)
Pr (X =l)
:
x
9e~®(l-X)
Pr (X.sJ) = Q^e'^/y. for
and
X
can be estimated from the sample as follows*
9 = £[x-l+*
+
{(x-l+m
2
)
(m -m 1 9 -1)
z
+4(x-m )}*] and
1
A
X
>2
2.71828 is the base of natural logarithms.
e =
9
J
1
(m +m.
For example, this becomes
1.174,
9 =
and for
X«_
r
Pr (XjSi)
=
X
s
3
4
>4
0.08
0.213
0.083
0.024
.008
4.0
10.7
4.2
1.2
0.4
1
.592
Pi = 29.6
Where Fl
= 0.78
2
Pr(X.=i) 50 is the expected frequency.
To test aroodness of fit, a chl-square crocedure is aDDlied:
,2
X
k
= E
In this test,
(
f4
the classes X^ and X>^ are lumped together so that
no class has an F,<1.
tests,
F.J with V degrees of freedom.
Therefore, V«5-3=2.
(In these chl -square
with
the degrees of freedom are always the number of classes
•
46
Pjjl less
3)
The chi -square value calculated is 11.82 which is sufficently
large to reject the hypothesis that the model fits the data observed.
The next step then is to eliminate the class X 1Q and repeat
the procedure.
Again, a sufficiently high chi-square value is
observed to reject the goodness of fit hypothesis.
Next,
the X^
class is eliminated along with all contiguous classes having an f^s 0.
This leaves
012 3^5
X 1=
=
37
Computing
s
t
t
5
.720,
X 1=
1
3
2
X s
.716, we find
12 3^
Pr (Xj«l) =
.738
.100
Pj =
35-4
4.8
.126
6.1
.030
1.5
.005
5
.001
.3
In order for the chi -square test to be unbiased,
classes are lumped together.
the X-, X^ and
X<-
The value of the test then is calculated
as 2.46 with one degree of freedom which is not large enough to
reject the hypothesis of goodness of fit, that is, the model is
adequate to describe this subset of the data.
Although a better fit
would result if the data were further truncated to say X~ it is
unnecessary.
Note that P(X,s5) <.001 and this is sufficient to adopt
the rule that any X>5 provides positive evidence of non randomness,
I.e.
colonization of Corynebacterium in the tracheal bifurcation.
^7
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BACTERIAL FLORA OF BOVINE RESPIRATORY TRACT
by
NICHOLAS AIGBEDO EVBUOMA
D. V. M., Ahmadu Bello University,
197^
An abstract of
A THESIS
.
submitted in partial fulfillment of the
requirements for the degree
MASTER OF SCIENCE
Department of Laboratory Medicine
KANSAS STATE UNIVERSITY
Manhattan, Kansas
197?
ABSTRACT
Bovine respiratory disease is a cause of great economic loss in the
cattle industry.
Its etiology has been attributed to a combination of complex
factors which include viruses, bacteria and environmental stress.
sp_.
Pasteurella
are the most frequently recovered bacterial isolate, but its association
with apparently healthy cattle lung tissue has not been reported.
The respiratory tract of 50 cattle were examined at slaughter for
aerobic bacteria.
Cotton tipped applicators were used to obtain tracheal and
bronchial fluids from ten locations in each lung.
Two locations in the trachea
and eight locations corresponding with the distal bronchi of the major pulmonary lobes were examined.
Four or five selective and differential media
were used for pixmary isolation.
A total of
1*33
isolates comprising 22 genera were recovered.
Streoto -
myces so. were the most frequently recovered and represented 22.756 of the
total isolates.
Pasteurella sp . represented
recovered from 18 lungs.
7.8?5 of
all isolates and were
They were considered transient in nine and coloni-
zing in nine.
The trachea and tracheal bifurcation were most frequently colonized by
bacteria.
There was no significant difference between levels of colonization
of different lobes of the lungs.
Pasteurella sn . colonized the trachea and
tracheal bifurcation more frequently than the bronchi.
It was hypothesized that
physical stress to which animals were subjected prior to slaughter may be
responsible for colonization of bronchi by pasteurellae.
It was concluded that the respiratory tract of apparently healthy beef
cattle was not a sterile environment.
The region from the trachea to the
distal bronchi were colonized by various species of bacteria, most of which
had their origin in the soil, feces or pharynx.
These organisms persisted
in spite of the elaborate mechanism by which the lung rids itself of particulate
matter.
This mechanism was not adequate to maintain sterility.
It was not
certain whether this Inadequacy was due to an inherent defect that was peculiar
to beef cattle or due to the presence of an overwhelming number of organisms
in inhaled air.
It is recommended that the pulmonary defense mechanisms in
feedlot cattle be further studied.
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