CAMPYLOBA.CTER FETUS SUBSPECIES JEJUNI FROM SOME COMMERCIALLY PROCESSED POULTRY PRODUCTS by Merton Vincent Smith II Thesis submitted to the Graduate Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Food Science and Technology APPROVED: b 1)?.' P. J. Muldoon I/ ....... ' Dr. M. D. Pierson Dr. R. M. Smibert
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CAMPYLOBA.CTER FETUS SUBSPECIES JEJUNI FROM SOME
COMMERCIALLY PROCESSED POULTRY PRODUCTS
by
Merton Vincent Smith II
Thesis submitted to the Graduate Faculty of the
Virginia Polytechnic Institute and State University in
partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
in
Food Science and Technology
APPROVED:
b 1)?.' P. J. Muldoon I/
~ ....... 1--~~~-----------h~-.._. ' Dr. M. D. Pierson Dr. R. M. Smibert
ACKNOWLEDGEMENTS
The author wishes to express his sincere appreciation to
Dr. P. J. Muldoon, Dr. M. D. Pierson and Dr. N. R. Krieg for their
suggestions, criticism and encouragement during the course of this
study and during the writing of this thesis.
Special thanks are given to Dr. R. M. Smibert for his tech-
nical advice and assistance.
Further appreciation is extended to Dr. J. Dekeyser, Dr.
R. E. Weaver and Dr. R. M. Smibert for supplying the Camwlobacter
fetus strains used in this study.
The author would like to thank the entire staff of the
Department of Food Science and Technology for their cooperation
and help.
ii
f?
TABLE OF CONTENTS
page
ACKNOWLEOOE~NTS · •••••••••••• • ••••••••••••••••••••••••••• ••'........ ii
REVIEW OF LITERATURE •••••••••••••••••••••••••••••••••••••••••••••• 3
Vibrio fetus Associated with Disease in Man ~
•••••••••••••••••• 3 Sources of Vibrio fetus in Animals Other than Birds ~ ••••••••••••••••••••••••••••••••••••••••••••••••••• 6 Sources of Vibrio fetus in Avian Species ••••••••••••••••••••• 9 Microflora of Processed Poultry Meat ••••••••••••••••••••••••• 11 Cultural and Isolation Methods for Vibrio fetus •••••••••••••• ~
~THODS AND MATERIALS ••••••••••••••••••••••••••••••••••••••••••••• 18
A Study of the Recovery of .Q.ampilobacter fetus from the Surface of Poultry Meat ••••••••••••••••••••••••••••• 18 A Study of the Survival of Campylobacter fetus on the Surface of Poultry Meat ••••••••••••••••••••••••••••••• 22 Survey of Retailed Poultry Meat and Liver •••••••••••••••••••• 24 Biochemical Characterization of Campylobacter ~Isolates ••••••••••••••••••••••••••••••••••••••••••••••• 24
RESULTS AND DISCUSSION ............................................ 27
Recovery Study ••••••••••••••••••••••••••••••••••••••••••••••• 27 Survival Study ••••••••••••••••••••••••••••••••••••••••••••••• 33 Survey of Retailed Poultry Meat and Liver •••••••••••••••••••• 38 Biochemical Characterization of Carnp:vlobacter ~Isolates ••••••••••••••••••••••••••••••••••••••••••••••• 41
Suggested Further Studies •••••••••••••••••••••••••••••••••••• 43
VITA ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••
iv
54
63
Tables in text:
Table 1.
Table 2.
Table 3.
Table 4.
Table 5.
Table 6.
Table 7.
Table 8.
Table 9.
LIST OF TABLES
page
Some differential characteristics of the species of the genus CampYlobacter ••••••••••••• 17
Strains of Campylobacter fetus used and their respective sources of isolation •••••••••• 21
Descriptive results of plating inoculated poultry meat utilizing various combinations of selective media and methods ••••••••••••••••••••• 28
Isolation of Camgy:lobacter fetus from inoculated poultry meat samples expressed as percentages of the total samples •••••••••••••••• 30
Identification of organisms commonly encoun-tered during the isolation of Campylobacter fetus from poultry meat •••••••••••••••••••••••••••• 32
Rec?very of Cam&lobact~r ~ during a period of 20 days storage at 3 C ••••••••••••• /...... 35
Recovery of Camp~lobacter fetus during a period of 20 days storage at -23.5°c ••••••••••••••• 36
Occurrence of CamEzlobacter fetus in some commercially processed poultry products •••••••e•••• 40
Some biochemical and morphological char-acteristics of strains of Campylobacter fetus ~· jejuqi and Campylobact2! fetus ss. intestinalis isolated from various sources •••••
Tables in appendix:
Table 10.
Table 11.
Table 12.
Analysis of variance of the type of organism used in the recovery study
Analysis of variance of the level of inoculum used in the recovery study
................
................ Detailed results of plating poultry meat inocul~ted with strain 7480 at a level of approximately lcY organisms utilizing various combinations of
55
selective media and methods •••••••••••••••••••••••• 57
v
Table 13.
Table 14.
Table 15.
Table 16.
Table 17.
LIST OF TABLES (CONT.)
page
Detailed results of plating poultry meat inoculated with ~train 7480 at a level of approximately 10 organisms utilizing various combinations of selective media and methods ••••••• 58
Detailed results of plating poultry meat inoculated with ~train 7480 at a level of approximately 10 organisms utilizing various combinations of selective media and methods ••••••• 59
Detailed results of plating poultry meat inoculated with strain B7988 at a level of approximately 103 organisms utilizing various combinations of selective media and methods ••••••• 60
Detailed results of plating poultry meat inoculated with gtrain B7988 at a level of approximately 10 organisms utilizing various combinations of selective media and methods ••••••• 61
Detailed results of plating poultry meat inoculated with 9train B7988 at a level of approximately 10 organisms utilizing various combinations of selective media and methods ••••••• 62
vi
LIST OF FIGURES
page Figure 1. Total surface aerobic bacterial count of
poultry meat during storage at 3°c and -23.5°c ••••••••• 37
vii
INTRODUCTION
Individuals trained in the field of veterinary science are familiar
with the_ bacteria Carnpylobacter fetus as a cause of bovine and ovine
abortion, avian hepatitis and scours in pigs and calves. Medical doctors,
in contrast, are generally not knowledgeable of these organisms as they
occur in human illness. Little interest in C. fetus has existed in the
medical comrrrunity to date primarily as a consequence of the small number
of reported human cases. Most investigators in this field believe that
the disease is highly underrated (Kilo et al., 1965; Bokkenheuser, 1968).
As a result of better surveillance and the use of appropriate isolation
techniques, a more representative incidence may be revealed.
The mode of transmission of human C. fetus infection is obscure
particularly when the disease is found in individuals living in an urban
environment without a history of animal contacts. Recently inferential
evidence has been presented that the orally transmitted, intestinal form
of C. fetus is associated with human vibriosis. White and Walsh (1970)
classified their C. fetus organisms according to the criteria of Laing
(1960). All of their human isolates were biochemically identical to the
C. fetus organism which is the etiologic agent of ovine vib~ionic abor-
tion and sporadic abortion in cattle. The vibrios associated with these
animal pathologies are known to be orally transmitted (Akkermans et~.,
1956; Bryans and Shephard, 1969).
The intestinal contents and various organs of some food animals
have been known to be sources of C. fetus. Many isolates from these
animals are morphologically, antigentically and biochemically indistin-
guishable from such isolates associated with human disease. It has been
1
2
suggested by several investigators that wild birds may be the vectors of
human vibriosis in the same way that they have been shown to be associated
with the disease in sheep.
The hypothesis of possible food-borne transmission of such inf ec-
tions in man forms the basis of this investigation. The purpose of this
study is to present evidence to indicate a possible food-borne epidemi-
ology for this human vibrionic infection. The research was undertaken
first to attempt to recover Q.• fetus ss. jejuni from commercially pro-
cessed chicken which was contaminated at various levels in vitro, then
to study the natural occurrence of such organisms in some retail
poultry meat and liver and finally to study the survival of selected
strains in poultry meat stored at refrigeration and freezing temperatures
over a period of several weeks. Retail poultry meat isolates were then
compared biochemically with isolates obtained from other sources.
REVIEW OF LITERATURE
VIBRIO FETUS ASSOCIATED WITH DISEASE IN MAN
Vinzent et al. (1950) reported the ~irst human infection of Vibrio
fetus. The case was that of a woman who had consumed milk from a cow
which had recently aborted. Since that time y. fetus infection in man
has become more commonly referred to in the literature; however, the
epidemiology still remains obscure.
Levy (1945) reported an outbreak of y. fetus gastroenteritis traced
to the milk supply. Vibrios were seen in the rrrucoid portion of the stools
and were cultured from the blood of several of the victims. King (1957)
presented a summary of human case histories. Of the 19 cases reviewed,
16 referred to previously existing chronic debilitative conditions
(primarily alcoholism and cardiovascular diseases). Digestive abnormal-
ities such as diarrhea and abdominal discomfort occurred in 12 cases.
King found no evidence to implicate food contamination as the epidemi-
ological vehicle in any of these cases. She felt that these vibrios
may be responsible for many of the cases of childhood diarrhea of
unknown etiology.
King (1962) in a later review of cases submitted to the Center for
Disease Control discussed one patient of significance with "related
vibrio" infection:
One was a chicken farmer who was an acute alcoholic with cirrhosis and hypertension. His case was of particular interest for several reasons. He had contact with chickens which are known to have a disease caused by a very similar, if not identical, organism. The patient became extremely dehydrated because of severe diarrhea and died in shock. At autopsy the jejunum and midway into the ileum were found to be hemorrhagic and congested, but there were no lesions
3
4
in the colon. This was the same type of pathology described by Jones et al. (1931) in cattle suffering from winter dysen-tery caused by Vibrio jejuni. One is tempted to conclude that the causative agents of avian infectious hepatitis, winter dysentery in cattle and vibrionic dysentery in humans are the same, although there is not yet sufficient proof.
King (1962) stated that the major symptom of y. fetus infection in
humans is diarrhea usually accompanied by blood and mucus. She felt
that the most significant work yet to be done is to discover the epidemi-
ology chain of events in these human vibrio infections. Bokkenheuser
(1969) compiled a more recent case review on human V. fetus infection.
He reported that 6 cases out of 57 exhibited no preexisting pathology
or abnormality. In the "related vibrio" infections the predominant
clinical feature was diarrhea. The primary sites of infection were the
blood and cerebral spinal fluid with no isolation or recovery from mixed
microflora in man.
The incidence of V. fetus infection in man is probably much under-
rated (Sp~, 1957; King and B~onsky, 1961; Kilo et~., 1965; Willis
and Austin, 1966; Bokkenheuser, 1969; White and Walsh, 1970). Bokkenheuser
(1969) believed that Jess than 1% of the cases are recorded. An indirect
bacterial hemagglutination test was employed by Bokkenheuser (1971) to
detect sero-reactors in infected or previously infected individuals. He
discussed the significance of the Laing (1960) intestinal form of y. fetus recovered from man by White and Walsh (1970) and by himself (1968):
All strains were recovered from mono-infected tissues. Conceivably the tissues were invaded from a site where the vibrios formed a part of a mixed flora, for example from the intestinal tract. The hypothesis that V. fetus could exist temporarily in the human gut offers an attractive explanation for the transmission of the organisms from animals and birds
5
to man, for the presence of low-titered I• fetus agglutinins in apparently noninfected individuals, and for the development of clinical infection in debilitated individuals.
Jacotot and Vallee"(1960) undertook a serological study of some
animals and men in France. Their results indicated that 90'/o of swine,
calves, sheep, monkeys and chickens contain antibod~es agglutinating
I• fetus. They found that 01/o of the infants tested, 20'/o of the children
and 30'/o of the adults showed positive sero-reactions. These results
suggest that man naturally comes into contact with microbes in the
environment which possess one or several antigenic fractions identical
to those of V. fetus • . -
Dolman (1957) reported I• fetus infection in a man with no occu-
pational exposure, b~t. who had consumed raw beef serum previous to the
infection. . He concluded that there is a potential hazard from ingestion
of the organism. Soonattrakul ~ ~· (1971) described the case of an
elderly woman with I• fetus ~· intestinalis septicemia apparently as
a result of ingesting raw beef liver as treatment for an anemic con-
dition. Mandel and Ellison (1963) described a case of acute I• fetus
dysentery syndrome in an otherwise healthy individual. On the basis
of prebacteriological findings a diagnosis of Salmonella or Shigella
gastroenteritis was made. Only through the recovery of a blood culture
was an accurate diagnosis possible. Recently Dekeyser et al. (1972)
recovered Y.• fetus from a mixed microflora (stool) in man by means of
a combination of selective filtration and subsequent culturing on
selective media. Their original isolates were obtained from the blood
and feces of a 22-year-old nurse with symptoms of bloody diarrhea,
6
nausea, sweating, fever, shivering and myalgia. In a subsequent survey
of 1000 stools from different individuals, Dekeyser tl ~· (1972)
obtained 35 strains of Y.• fetus from two adults and 28 children. Nine-
teen of these children and both adults showed diarrheic symptoms. These
investigators felt that there may be a great many cases of diarrhea
caused by "related vibrio", but thus far are undetected because of an
unconciousness of their possible presence and the use of inadequate
bacteriological methods to isolate the organism.
There seem to be no differences either morphologically, bio-
chemically (Smibert, 1969) or antigenically (Sibinovic, 1965; Dekeyser
tl ~., 1972) between many "related vibrios" of man and V. fetus of
poultry origin. Fletcher and Plastridge (1964) noted average differences
between five chicken strains of V. fetus and five human strains. They
concluded that avian strains exhibited the following characteristics:
a shorter survival time when grown in thiol medium, a lower tolerance
for sodium chloride, capability of growth at 45°c and significant
differences in deaminase activity when compared to the human strains.
SOURCES OF VIBRIO FETUS FROM ANIMALS OTHER THAN BIRDS
Vibrio infection was first recognized by McFaydean and Stockman
(1909) in England. They associated it with abortion in cattle and
sheep. The organism was isolated, named and studied for the first time
by Smith (1918). Infectious vibrionic abortion of cattle is a venere-
B. 15 units bacitracin/ml, 5 )Jg novobiocin/ml, 5 units polymyxin/ml.
c. 15 units bacitracin/ml, 5 pg novobiocin/ml, 1 unit polymyxin/ml.
D. 5 units bacitracin/ml, 5 µg novobiocin/ml, 1 unit polymyxin/ml.
E. 5 units bacitracin/ml, 2 )4g novobiocin/ml, 1 unit polymyxin/ml.
F. 2 units bacitracin/ml, 2 )Ag novobiocin/ml, 1 unit polymyxin/ml.
G. 0 units bacitracin/ml, 0 )Ag novobiocin/ml, 0 units polynzyxin/ml.
The unfiltered nutrient broth wash was swabbed onto three plates of
brucella agar media containing each of the following:
A. 15 units bacitracin/ml, 15 µg novobiocin/ml, 5 units polymyxin/ml.
B. 15 units bacitracin/ml, 5 }Af, novobiocin/ml, 5 units polymyxin/ml.
c. 2 units bacitracin/ml, 2 )J.g novobiocin/ml, 1 unit polymyxin/ml.
D. 0 units bacitracin/ml, o~ novobiocin/ml, 0 units polymyxin/ml.
Antibiotic solutions were prepared by dissolving bacitracin, nova-
biocin and polynzyxin B sulfate in phosphate buffer (0.02 M Na2HPo4-KH2Po4)
to obtain concentrations which could be added conveniently to the basal
agar medium. These stock solutions were filter sterilized and frozen at
-23°c until use.
All plates were incubated under microaerophilic conditions at 37°c
for 5 days and then qualitatively evaluated and classified according to
the following plate description categories:
A. C. fetus isolated.
B. No C. fetus isolated, other isolated colonies present.
C. No visible colonies present.
D. Plate completely overgrown.
A STUDY OF THE SURVIVAL OF CAMPYLOBACTER FETUS ON THE SURFACE OF POULTRY MEAT
Thirty-two samples of chicken necks were collected and held for
23
use in the same manner as those of the preceding RECOVERY STUDY. All
samples were utilized within 10 days after being obtained at the pro-
cessing plant.
The samples were split aseptically as previously discussed. Total
surface aerobic plate counts of each control subsample were undertaken
by the standard swab-rinse method used in the RECOVERY STUDY. Isolatio~
of any preexisting £• fetus present was attempted by plating unfiltered
nutrient broth poultry meat wash on the surfaces of two plain brucella
agar plates and two brucella agar plates combining 15 units bacitracin/
ml, 15,,Mg novobiocin/ml and 5 units polymyxin/ml. Approximately 3-4 ml
of filtered nutrient broth wash was swabbed onto the surface of 3 plain
brucella agar plates and on the surface of brucella agar plates incor-
porating 2 units bacitracin/ml, 2JAf, novobiocin/ml and 1 unit poly-
myxin/ml until the filtrate was completely plated. 6 One-tenth percent peptone suspensions of approximately 10 organ-
isms were then inoculated on the surface of the remaining 32 subsample
poultry neck halves. The organisms used were £• fetus strains 29A
(poultry meat isolate) and 7480 (chicken liver necropsy isolate).
Inoculated subsamples were stored in 18 ounce polyethylene bags at 0 0 temperatures of 3 C and -23.5 C. A subsample of each type of inoculum
was removed from storage after the following times: 0 days, 5 days,
10 days and 20 days. Frozen subsamples were allowed to thaw before
testing •. Standard surface plate counts were calculated as previously
described.
Recovery of C. fetus microorganisms was attempted in the same
manner as outlined for the control subsamples.
24
SURVEY OF RETAILED POULTRY MEAT AND LIVER
One hundred and twenty-one chicken necks were purchased from 5
local retail food markets and one local poultry processing plant.
Quantities obtained for sampling ranged from an entire pack of 23 necks
to packages from which only one sample was taken. Twenty-five chicken
livers were purchased from 2 local retail markets. Samples varied from
a group of 5 livers in the same package to individually sampled packages.
Nineteen whole dressed broilers were obtained from 2 local food stores
all of which were individually packaged.
A standard surface aerobic count was taken on each type of sample
by the swab-rinse method (Sharf, 1966) to determine the general level
of microbial contamination. Counts on the whole broilers were deter-
mined by swabbing 2 cm2 of the back portion and 2 cm2 of the breast
area and plating each in duplicate. The total counts for the liver
samples were derived by diluting the blended liver and counting colonies
on pour plates as described by Sharf (1966).
Detection of possible .Q.• fetus present was undertaken as in the
SURVIVAL STUDY previously described except for the following variations
in procedure: whole chickens were placed in 128 ounce sterile poly-
ethylene bags and thoroughly washed with 50 ml of nutrient broth over
the entire exposed surface. Each chicken liver was blended with 50 ml
nutrient broth. Ten grams of this mixture was then plated before and
after filtration (0.65jlm) as discussed in the RECOVERY STUDY.
BIOCHEMICAL CHARACTERIZATION OF CAMPYLOB\CTER FETUS ISOIATES
· Strains· of C. fetus ~· JeJuni isolated from poultry meat were
25
compared to strains of Q.• fetus .2.§.• jejuni and Q.• fetus .2.§.• intestinalis
obtained from other sources described in Table 2.
Biochemical tests were performed according to the methods of
Smibert (1972). Glucose fermentation was determined by acid formation
in semi-solid base broth containing 1% glucose and 0.002% phenol red
indicator. Nitrate and nitrite reduction were indicated by standard
tests after growth in semi-solid base broth with 1% KN03• Positive or
negative growth in semi-solid base broth plus 3.5% NaCl and semi-solid
base broth plus 1% glycine were determined. Hydrogen sulfide production
was detected by growth on SIM medium (Difeo Laboratories, Detroit, Mich.)
and by growth in semi-solid broth base containing 0.02% cysteine hydro-
chloride (Fisher Scientific, Raleigh, N. C.) and a lead acetate test
paper (Fisher Scientific, Raleigh, N. C.) resulting in a slight brown
to dark black discoloration after incubation at 37°c for 10 days.
Growth or no growth in semi-solid broth base plus 1% bile salts (Dif co
Laboratories, Detroit, Mich.) was indicated. Temperature tolerance
tests were determined by growth in semi-solid base broth at 25°c, 42°c and 45°c. Incubation at 25°c was carried out in a standard air convection
type incubator, and incubation at 42°c and 45°c was done in a New
Brunswick R77.Water Bath Shaker (New Brunswick Scientific Co., Inc.,
New Brunswick, N. J.). Brucella agar plates were streaked and incubated
under each of the following atmospheres: microaerophilic (anaerobe jar
replaced with 5% o2, 1o% co2, 85% N2); anaerobic [Gas Pak jar (BBL,
Cocke~sville, Md.)]; and aerobic. Growth was indicated as either pos-
itive, negative or weak. Oxidase production was detected by use of
Taxo N phenol oxidase test discs (BBL, Cockeysville, Md.) on the micro-
26
aerophilic plate. Catalase production was indicated by bubble formation
within 1 to 2 minutes following the addition of 1 ml of 3% H2o2 solution
to the growth in semi-solid base broth.
All tests were carried out on cultures incubated for 7 days at
37°c except where indicated otherwise. All semi-solid base broth media
were dispensed about 3 inches deep (10 ml) in 16 X 150 mm screw cap
tubes. The stock cultures were maintained by transferring every two to
three days and then weekly after the organisms demonstrated good growth
on the artifical medium.
Viability and pure culture were constantly surveyed by phase
contrast microscopic examination. All broth media containing. 0.1&%
agar were incubated in normal atmospheric condit,ions, while solid agar
media were incubated under 5% o2, 10 % co2 and 85% N2 except where
specified to the contrary.
RESULTS AND DISCUSSION
RECOVERY STUDY
Total aerobic plate count for the 30 poultry meat samples exhibited
an average geometric mean of 5.1 X 104 organisms/cm2 and a range from
3.3 X 1o3 organisms/cm2 to 2.1 X 105 organisms/cm2• Campylobacter fetus
or microaerophilic vibrios were not detected on the control subsample
halves when using the BN selective agar and selective filtration employed
by Smibert (1964a, 1972). This indicated that prior to the 1!! vitro
inoculation these samples were not naturally contaminated with vibrios
at a level sufficiently high to be detected by these methods.
After diluting and plating one milliliter of each inoculum level,
it was determined that the following number of viable organisms were
used as the inocula for the recovery study:
Strain 7480 ~ 5.2 X 103, 4.6 X 106, 4.0 X 109 organisms/ml.
Strain B7988~ 5.5 X 103, 5.4 X 106, 5.8 X 109 organisms/ml.
Table 3 presents a qualitative descriptive summary of the recovery
results as precentages of the total plates used for each selective
medium and method at all levels of inoculation and for both strains
used as inocula.
The concentrations of selective antibiotics employed by Shepler
et al. (1963) and Plastridge et al. (1961) were used in this study as ~ ~ ~ ~
maximum and minimum parameters respectively. Within this range it was
found by this author that the greatest recovery of the two strains of
Camwlobacter, based on the percentage of the total plates used, was
by selectively filtering and plating on agar incorporating bacitracin
27
Table 3. Descriptive results of plating inoculated poultry meat utilizing various combinations of selective media and methods.
PLATE DESCRIPTION ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F'
Q• fetus isolated 36* 33 24 33 41 46 11 46 9 4
No C. fetus isolated 4 4 33 29 (other isolates present)
40 13 27 38 86 54
No visible colonies on 59 61 42 38 19 41 40 6 4 0 plate
Plate completely 1 0 0 0 0 0 22 11 1 44 overgrown
*each value is expressed a~ a percentage of the total plates used for each selective method.
A = 15 units bacitracin/ml~ B = 15 units bacitraciri/m1 7 C = 15 units bacitracin/ml, D = 5 units bacitracin/ml, E = 5 units bacitraciri/ml, F ~ 2 units bacitracin/ml, G = 0 units bacitracin/ml, ' = without filtration.
15 _µg novobiocin/ml~ 5 units polynzyxin/ml. 5 }.cg novobiocin/m1 7 5 units polynzyxiri/ml. 5 ;cg novobiocin/m1 7 1 unit polynzyxiri/ml. 5 ;cg novobiocin/ml, 1 unit polynzyxiri/ml. 2 >'g novobiocin/ml, 1 unit polynzyxin/ml. 2 .>Cg novobiociri/ml, 1 unit polynzyxiri/ml. O JAg novobiocin/ml, O units polynzyxiri/ml.
G'
0
22 I\) 00
2
76
29
at a level of 2 units/ml, novobiocin at a level of 2µg/ml and polynwxin
at a level of 1 unit/ml (level F in Table 3).
The results of the recovery study were also analyzed on the basis
of the total samples used and were compiled as shown in Table 4 (de-
tailed data are presented in Tables 12 to i7 of the Appendix). Highest
isolation of C. fetus based on per sample data was also at antibiotic
combination F. Total recoveries at the next highest level of these
A= 15 units bacitracin/ml, 15µg novobiocin/m1 7 5 units polymyxin/ml. B = 15 units bacitracin/m1 7 5 ;.tg novobiocin/m1 7 5 units polymyxiri/ml. C = 15 units bacitracin/m1 7 5 µg novobiocin/ml, ~ unit polymyxin/ml. D = 5 units bacitracin/m1 7 5 µg novobiociri/m1 7 1 unit polymyxiri/ml. E = 5 units bacitracin/ml, 2 f(g novobiocin/ml, 1 unit polymyxin/,ml. F = 2 units bacitracin/ml, 2 p.g novobiocin/ml, 1 unit polymyxiri/ml. G = 0 units bacitracirl/ml, 0 ;-tg novobiocin/ml, 0 units polymyxin/ml.
= without filtration.
31
3. Polymyxin was utilized for samples in this survey to inhibit
the possible growth of Campxlobacter sputoru.!!l•
There was a higher percentage of £• fetus isolations from media
combining the higher levels of antibiotics when no filtration was used.
For this reason agar media with 15 units bacitracin/ml, 15;.<g novo-
biocin/ml and 5 units polymyxin/ml was employed in this survey for
samples which were not filtered.
It was noted that there was no significant difference (cC.= 0.025)
between the percentages of isolations from filtered samples plated on
media combining the optimal levels of antibiotics and samples unfiltered
and directly surface plated on media combining the optimal levels of
antibiotics. However, the filtered samples contained fewer contaminating
colonies of Q. fetus morphology. In general, the filtered samples re-
sulted in a more rapid £• fetus isolation with fewer organisms picked.
The unfiltered plates often contained many small colonies of organisms
other than C. fetus (e.g. Proteus, Pseudomonas, Alcaligenes) as identi-
fied in Table 5.
There has been no differential medium developed for distinguishing
C. fetus from contaminating organisms. Contaminants were the most
troublesome problem in the isolation and recovery of C. fetus from mixed
culture. The sele~tive methods of filtration and agar combining anti-
biotics to which£· fetus is relatively insensitve make its isolation
possible; however, certain organisms commonly found on poultry such as
slender Alcaligenes (0.5 by 1.0 to 2.0fAm) are capable of penetrating
a 0.65 fAm filter. Also common contaminants such as Proteus and §.• ~'
which would normally spread over the plate or form extremely large
32
Table 5. Identification of organisms commonly encountered during the isolation of Carnpylobacter fetus from poultry meat.
*identified by API Analytab System (Analytab Products Inc., New York)
33
colonies after incubation at 37°c for 3-5 days, form small 1-2 mm colonies
often indistiguishable from those of .Q.. fetus. These objections make it
impossible to quantitatively determine survival or recovery from a
natural mixed microflora such as that of poultry meat.
Alcaligenes faecalis was a particularly annoying contaminant. This
organism can be easily mistaken for f • fetus after the initial screening
procedures described earlier. It does not ferment glucose, is relatively
thin, produces catalase and oxidase, is motile and often exhibits a
curved rod form.
There were no significant differences in the percentages of iso-
lations between the two types of organisms used or among the levels of
these inoculums based on an analysis of variance of the data at a 0.05
significance level (see Tables 10 and 11 of the Appendix). The author
is unable to explain the similar recovery for all three levels of ino-
culum. This further emphasizes the difficulty that would be encountered
if a quantitative recovery were attempted.
SURVIVAL STUDY
Total surface aerobic counts of the control subsamples ranged from
7.5 X 103 organisms/cm2 to 2.2 X 105 organisms/cm2 with a geometric
mean of 5.6 X 104 organisms/cm2•
After diluting and plating one milliliter of each inoculum type,
it was determined that 3.8 X 106 organisms of strain 29A and 4.3 X 106
organsims o'f strain 7480 had been inoculated on the surface of the
survival subsample halves.
Recovery of viable Q• fetus after O, 5, 10, 20 days is shown on
34
Tables 6 and 7 after storage at 3°c and -23.5°c respectively. Detection 0 of Q. fetus after 5 days at 3 C was accomplished for inoculum strain
29A while no isolates of strain 7480 inoculum were obtained after 5 days
at this temperature. There were no isolates of either type detected
after 10 or more days at 3°c. All samples after 10 days storage at
3°c exhibited a distinctive spoilage odor, and after 20 days a slimy
surface appearance was also evident. Strain 29A survived and was de-
tected after O, 5, 10 and 20 days of frozen storage. Strain 7480
organisms were detected at O, 5 and 10 days at -23.5°c.
Total counts for each storage period and temperature are illu-
strated in graphic form in Figure 1. These. data are the arithemic 2 means of 3 separate template surface swab samples of 2 cm each. The
data compare well with the results of Essary~~· (1960) and Ayres
et al. (1950).
The isolation of viable Q• fetus from poultry meat after 5 days
storage at 3°c is quite significant since fresh poultry is commonly not
held for a longer period. In fact, the poultry meat sampled after 10
days storage would be considered spoiled by some investigators on the
basis of spoilage odor (Essary~~., 1958).
The results of this study are comparable to those of Peckham (1958)
who found that the organism remains viable in avian tissues and bile
stored at 4°c for 6 days.
The study of Lindenstruth and Ward (1948) also suggested that a
lowered temperature results in a longer survival of the organism. They
report survival for 20 days at 6°c. Perhaps a reduced psychrophilic
population in the mixed cultures or the greater inoculum level of
35
Table 6. Recovery of Campylobacter fetus during a period of 20 days storage at 3°c.
STRAIN NUMBER 0
29A + 29A (duplicate) + 7480 + 7480 (duplicate) +
+ = positive isolation of Q• fetus - = no isolation of c. fetus
TIME (days) 5 10 20
+
Table 7. Recovery of Camp~lobacter fetus during a period of 20 days storage at -23.5 C.
STRAIN NUMBER
29A 29A (duplicate) 7480 7480 (duplicate)
O*
+ + + +
+ = positive isolation of Q• fetus - = no isolation of C. fetus * frozen for 45 minutes before sampling
TIME (days)
5
+
+ +
10
+
+
20
+
37
9
8
7 C\l :?! u .........
6 a:: w (]) 5 :E ::::> z 4 {!) 0 _J 3
6 3°C 2 0 -23.5° c
0 0 5 10 15 20
STORAGE TIME (DAYS)
Figure 1. Total surface aerobic bacterial count of poultry meat during storage at 3°c and -23.5°c.
38
of Q• fetus (1.5 X 109) resulted in the longer survival in their study
as compared to this author's findings.
Recovery in terms of ratio of aerobic organisms to C. fetus is
much lower than compared to the results of Bokkenheuser (1968). It is
probable that the storage time involved in the current study lessened
the number of viable C. fetus from the original 106 organisms to a
minimal number. It is not known what type of medium was filtered by
Bokkenheuser. No doubt the surface fat of chicken along with tissue
pieces serve to clog the filtering apparatus in recovery from poultry
meat.
The survival of Q• fetus during storage at -23.5°c indicates
that the organism will remain viable on poultry meat if it is frozen
at this low temperature. It can be predicted based on the observations
of Smibert (1973) that if the poultry meat were frozen at an even
lower temperature viability of the organism and subsequent recovery
would be enhanced. Smibert (1973) has found that in freezing stock
culture collections the survival of such pure cultures is extended by
several years when the freezing temperature is lowered from -30°C to 0 -85 c.
If the ingestton of viable C. fetus organisms proves to be a
human health hazard, the results of this survival study suggest that
the greatest danger of infection in poultry meat products is from the
fresh (5 days or less after processing) or frozen product.
SURVEY OF RETAILED POULTRY MEAT AND LIVER
A summary of the survey of the incidence of C. fetus in poultry
meat products purchased from 5 local groceries and a local processing
39
plant is presented in Table 8. All of these samples were purchased in
a refrigerated state and underwent immediate bacteriological analysis
upon receipt in the laboratory.
The average counts on the back and breast areas of the whole
broilers were 6.0 X 104 organisms/cm2 and 2.4 X 104 organisms/ cm2
respectively. Two £. fetus .§.2.• jejuni isolates (strains C14 and 29A)
were obtained from the surfaces of 121 chicken necks sampled for a
1.7% incidence. These samples included the 62 chicken neck control
subsamples from the SURVIVAL and RECOVERY STUDIES. One isolate (strain
K14C) was found on the 19 whole broilers for a calculated 5.3% inci-
dence. No isolates were detected in the liver samples.
The total counts for the chicken necks containing Q• fetus were
5.2 X 104 and 7.5 X 104 organisms/cm2• There was no significant
difference (CX:.= 0.05) between these counts and the average overall
count for neck surface organisms --5.5 X 104 organisms/cm2• There
was also no significant difference (CC= 0.05) between the breast surface
total count (1.9 X 104 organisms/cm2) on the whole broiler from which
a C. fetus organism was isolated and the average breast surface count
for all 19 whole broilers (2.2 X 104 organisms/cm2).
Difficulty of isolation and the results of such studies as
Reiland and Hurvell (1970) indicating the magnitude of loss through
filtering the vibrios suggest that the incidence of C. fetus as found
in the SURVEY OF RETAILED POULTRY MEAT AND LIVER is a minimum value.
Since these results reflect an extremely low incidence of Q• fetus
organisms in the poultry products sampled, the possibility that these
isolates were laboratory contaminants is discussed. The author con-
40
Table 8. Occurrence of Campylobacter fetus in some conunercially processed poultry products.
TYPE OF POULTRY SAMPLE
Chicken Necks
Chicken Livers
Whole Dressed Broilers
Total Poultry Meat (not Livers) ·
Total Poultry Samples
*geometric means
NUMBER OF SAMPLES
121
25
19
140
**total aerobic count per gram
TOTAL AEROBI~ COUNT PER CM *
5.5 x 104
2.0 x 105**
2.7 x 104
4.7 x 104
c •. fetus ISOIATi'ONS
No. Percent
2
0 0
1
3
3
41
siders that such a possibility is unlikely. The first Q• fetus poultry
meat isolate (strain 29A) was isolated, cultured and identified before
any reference strains were in the laboratory area. In addition,
standard aseptic bacteriological techniques were employed at all stages
of this investigation.
BIOCHEMICAL CHARACTERIZATION OF CAMPYLOBACTER FETUS'ISOIATES
A summary of the biochemical comparison of Q• fetus isolates
obtained from retailed poultry meat and Q• fetus organisms from other
sources is presented in Table 9. It should be noted that isolates
653, B6455, B7988 and B8615, unlike the others, grow at 25°c and not
at 45°c and are, therefore, classified as C. fetus ss. intestinalis.
With slight differences, the remaining Q• fetus ~· jejuni isolates are
biochemically identical on the basis of these characteristics (Table 9).
There were no demonstrable biochemical differences among the
human"related vibrios" and poultry isolates of all types.
·The results reported by Fletcher and Plastridge (1964) suggest
differences in human "related vibrio" strains and avian vibrionic
hepati:Hs strains. It must be noted that their results are simply
average findings based on five strains from each source. Several human
strains are practically identical to some avian strains even on the
basis of the tests used by Fletcher and Plastridge. As reported in
Table 9, human strains H718, H550, H840, H563, B7619 grow as well as
or better than the avian strains at 45°c.
It must be emphasized that many of the "related vibrios" of human
infection are indistinguishable from avian vibrios on the basis of
morphological, biochemical and antigenic tests.
42
Table 9. Some biochemical and morphological characteristics of strains of Camg{lobacter fetus ~· jejuni and CampYlobacter fetus ~· intestinalis isolated from various sources.*
TEST OR CHARACTERISTIC
glucose fermentation •••••••• N03 reduction •••••••••••••• N02 reduction •••••••••••••• 1% glycine growth •••••••••• 3.5% NaCl growth ••••••••••• H2S on SIM ••••••••••••••••• HAS on 0.02% cysteine**••••• 1~ bile growth ••••••••••••• oxidase •••••••••••••~•••••• catalase ••••••••••••••••••• motility ••••••••••••••••••• aerobic growth ••••••••••••• anaerobic growth ••••••••••• 5%002 growth ••••••••••••••• 25 C growth •••••••••••••••• 42~C growth •••••••••••••••• 45 C growth ••••••••••••••••
postive test or growth• negative test or growth. weakly positive test or growth.
*for organism identity and reference of the various sources see Table 2. ** detected by Difeo test strips (PbAc) after 10 days incubation. *** detected by BBL phenol oxidase differentiation discs. ****this culture died soon after.identification (tested only once).
43
SUGGESTED FURTHER STUDIES
The significance of these findings cannot be evaluated until further
studies are undertaken. Such future investigations could include an
attempt to infect domestic foul with human strains of "related vibrio" to
determine the interspecies virulence potential of these organisms. The
incidence of£• fetus in products of other food animals (e.g. pork,
turkey, duck, lamb, mutton, beef) should be determined. Information on
the effects of human ingestion of live £• fetus organisms must be gath-
ered to determine the importance of such bacteria in food. The in-
gestion of these bacteria may produce immediate gastrointestinal distress,
or it may simply establish the organism in the intestinal tract in low
numbers later to cause illness when the individual's resistance is low.
CaIBPYlobacter fetus may simply be a normal inhabitant of the human
digestive tract as Soonattrakul (1971) has suggested. The question of
how the organism is initially established as part of the digestive flora
is of no less _importance.
If, after further research on the epidemiology of£• fetus infec-
tion, it is concluded that the organism Imlst be eliminated from food
products; it would prove to be difficult for the poultry industry.
Culling for diseased birds is impossible. The major symptom_ in the
chicken is a "general unthriftiness" (Truscott and Stockdale, 1966).
Only a small p~rcentage of the birds show any clinical signs of disease
(Sevoian ~ ~., 1958).
The implica~ions of £• ~ present in food products may be of
special signif:i,ca;nce inthepreparation of food for debilitated indi-
viduals such as in a nursing home or hospital environment. Stress
factors such as liver fluke infestation (Hofstad~~., 1973) irt chick-
ens or pregnancy and harsh climatic conditions irt sheep (Winkenwerder
and Maciak, 1964) appear to be an important precondition for the establish-
ment of vibrionic disease irt these animals. Secondary infections are
becoming irtcreasirtgly important in such human diseases as cancer or irt
transplant patients on imnrunosuppressive therapy. An additional concern
to clinicians is the appearance of antibiotic resistant vibrios ·
(Bokkenheuser, 1969).
A realistic perspective of the importance of these organisms irt
human pathology will come about only through an awareness of the presence
of such bacteria or the possibility of encountering them during the
bacteriological analysis of certairt foods and clinical specimens from
man.
SUMMA.RY
A method for the recovery of Campylobacter fetus from poultry meat
using known selective methods was described and utilized to isolate the
organism from the surface of chicken necks contaminated 1.!1 vitro and
from retailed commercially processed poultry products.
One C. fetus strain was capable of surviving for 5 days on the
surface of poultry meat stored at 3°c and for 20 days when stored at 0 0 -23.5 C. Two strains survived on the meat surface for 10 days at -23.5 c. The incidence of £· fetus in 165 samples of retail poultry meat and
liver was found to be 1.8%. All three £• fetus colonies isolated were
recovered initially from selective brucella agar plates (2 units of
bacitracin/ml, 2,µ.g of novobiocin/ml and 1 unit of polymyxin/ml) which
had been swabbed with a filtered nutrient broth wash from the poultry
sample. Two of these isolates were from the surface of chicken necks
and one was isolated from the surface of a whole dressed broiler. No
significan:t differences existed between the average total surface counts
of the £• fetus contaminated poultry meat samples and similar samples
from which no c. fetus was isolated.
The C. fetus ss. jejuni strains isolated from commercially pro-
cessed chicken meat were biochemically and morphologically indistin-
guishable from other Q• fetus ss. jejuni strains isolated from human
and avian disease.
Campylobacter fetus organisms are capable of surviving on the
surf ace of chicken at refrigeration and freezing temperatures for a
period sufficently long to allow the meat to be marketed at the retail
45
level. In addition, such organisms have been detected on chicken meat
purchased through retail distributors. The significance of these find-
ings cannot be determined without further investigations concerning the
human infectivity of such organisms.
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Vinzent, J •• Delarue, J. and Herbert, H. 1950. L'infection placentaire ~ Vibrio foetus. Ann, Med. 51:23-68.
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Walker, H.W. and Ayres, J.C. 1956. Incidence and kinds of microorganisms associated with commercially dressed poultry. Appl. Microbiol. 4:345-349.
53
White, F.H. and Walsh, A.F. 1970. Biochemical and serologic relation-ships of isolants of Vibrio fetus from man. J. Infec. Dis. 121: 471-474·
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APPENDIX
54
55
Table 10. Analysis of variance of the type of organism: used in the recovery study.
SOURCE OF VARIATION
Type of organism
Selective medium or method
Residual
Total
SS
0.2
391.8
13.8
405.8
df
1
10
10
21
MS F
0.20 0.144
39.20 28.400
1.38 - -- --
Table 11. Analysis of variance of the level of inoculum used in the recovery study.
SOURCE OF VARIATION
Level of inoculum
Selective medium or method
Residual
Total
SS
0.8
262
36.7
299.5
df
2
10
20
32
MS F
0.40 0.22
26.20 14.20
1.84 --- -
Table 12. Detailed results of plating poultry meat inoculated with strain 7480 at a level of approximately 103 organisms utilizing various combinations of selective media and methods.
SAMPIE FLA.TE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F' G'
1 A NGR NGR ISO NIC NIC ISO OGR NGR ISO OGR OGR B NGR ISO NGR NGR NGR NGR NGR ISO OGR OGR OGR c NGR NGR ISO NGR NIC NIC OGR ISO NGR ISO OGR
2 A NGR NGR NGR ISO NIC ISO OGR NIC NIC NIC OGR B ISO NGR NGR NIC NIC NGR NGR OGR NIC OGR OGR c NGR NGR NGR ISO NIC ISO NGR NIC NIC NIC OGR
3 A ISO ISO NIC NGR ISO NIC ISO ISO NIC NIC OGR B ISO NGR NGR NIC ISO ISO NIC NIC NIC NIC OGR c NGR NGR NIC NIC ISO NGR NIC NIC NIC ISO OGR
4 A NGR ISO NGR ISO ISO ISO NIC NIC NIC NIC NIC \Jl -..J
B NGR ISO ISO NIC ISO ISO NGR OGR NIC OGR OGR c NGR NGR NIC NGR NGR NGR NGR ISO NIC OGR OGR
5 A NGR ISO NIC ISO NIC NGR ISO ISO NIC NIC NIC B NGR NGR NIC ISO NIC NGR NGR NIC NIC OGR OGR c NGR NGR ISO NGR ISO NGR NIC ISO ISO NIC OGR
OGR = completely overgrown plate, ISO = isolated £• ~tus, NIC = no isolated £• fetus, but isolated contaminant colonies present, NGR = no colonies presenr-(no growth). A = 15 units bacitracin/ml, 15,,ug novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5µ.g novobiocin/ml, 5 units polymyxin/ml. C = 15 units bacitracin/ml, 5µg novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitracin/ml, 5,µg novobiocin/ml, 1 unit polymyxin/ml. E -= 5 units bacitracin/ml, 2 }J..g novobiocin/ml, 1 unit polymyxin/ml. F = 2 units bacitracin/ml, 2)-'g novobiocin/ml, 1 unit polymyxin/ml. G = 0 units bacitracin/ml, O;-<g novobiocin/ml, 0 units polymyxin/ml.
= without filtration.
Table 13. Detailed results6of plating poultry meat inoculated with strain 7480 at a level of approximately 10 organisms utilizing various combinations of selective media and methods.
SAMPLE PLATE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F' G'
6 A ISO ISO NIC ISO ISO ISO OGR OGR NIC OGR OGR B NGR NGR NIC ISO NGR ISO NIC ISO NIC OGR NIC c ISO NGR ISO ISO NIC NGR OGR ISO NIC NIC OGR
7 A NGR NGR ISO NIC ISO ISO NIC NIC NIC OGR OGR B ISO NGR ISO ISO ISO NGR NGR NIC NIC NIC OGR c NGR NGR NGR NIC NGR NIC OGR NIC NIC NIC OGR
8 A NGR ISO NIC NGR ISO NGR NGR ISO ISO NIC OGR B NGR NGR NGR NIC ISO ISO NGR ISO NIC NIC OGR c NGR ISO NGR NGR NIC NGR NGR ISO NIC OGR NIC
9 A ISO ISO NIC ISO ISO ISO OGR NIC NIC OGR OGR \JI ~
B ISO NGR NGR ISO ISO NGR NIC ISO NIC OGR OGR c ISO ISO ISO NGR NIC NGR NIC NIC NIC OGR NIC
10 A ISO ISO NGR NIC NGR ISO OGR NGR NIC NIC NIC B ISO NGR NGR NGR NIC NGR NGR ISO NIC NIC OGR c NGR NIC NGR NGR NIC ISO NIC ISO NIC NIC NGR
OGR = completely overgrown plate, ISO = isolated£• fetus, NIC = no isolated£• fetus, but isolated contaminant colonies present, NGR = no colonies present (no growth). A = 15 units bacitracin/ml, 15 pg novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5Pg novobiocin/ml, 5 units polymyxin/ml. C = 15 units bacitracin/ml, 5 )Ag novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitracin/ml, 5.JAg novobiocin/ml, 1 unit polymyxin/ml. E = 5 units bacitracin/ml, 2 _µ.g novobiocin/ml, 1 unit polymyxin/ml. F = 2 units bacitracin/ml, 2 ~g novobiocirl/ml, 1 unit polymyxin/ml. G = 0 units bacitracin/ml, OJ-lg novobiocin/ml, O units polymyxin/ml. ' = without filtration.
Table 14. Detailed results of plating poultry meat inoculated with strain 7480 at a level of approximately 109 organisms utilizing various combinations of selective media and methods.
SAMPLE PLATE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c n E F G A' B' F' G'
11 A NIC NGR NGR NGR NIC NGR NGR ISO NIC NIC NGR B ISO NGR NGR NGR NIC NGR NGR NIC ISO NIC OGR c NGR NGR NGR NIC NIC ISO NGR NIC NIC NIC NIC
12 A ISO NGR NIC NGR NIC ISO OGR OGR NIC OGR OGR B NGR NIC ISO NIC ISO ISO NIC NIC NIC OGR OGR c ISO NGR NGR NGR ISO NGR OGR NIC NIC NIC OGR
13 A ISO ISO ISO NGR NIC ISO ISO ISO NIC OGR OGR B NGR ISO NIC ISO ISO ISO NGR ISO , NIC OGR NIC c ISO NGR NIC ISO ISO ISO NIC NIC NIC NIC OGR
14 A NGR NGR NIC NGR NGR NGR ISO NIC NIC OGR OGR \.Tl
'° B ISO NGR NGR ISO ISO ISO NGR ISO NIC NIC OGR c NGR ISO NIC ISO ISO ISO OGR NIC NIC OGR NIC
15 A NGR NGR NGR NGR NGR ISO NIC ISO NGR NIC OGR B NGR NGR NGR NIC NGR NGR NGR NGR ISO NIC OGR c NGR NGR NGR NGR ISO ISO OGR ISO NIC OGR OGR
OGR = completely overgrown plate, ISO = isolated£• £etus, NIC = no isolated£• fetus, but isolated contaminant colonies present, NGR = no colonies present (no growth). A = 15 units bacitracin/ml, 15).(g novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5 pg novobiocin/ml, 5 units polymyxin/ml. C = 15 units bacitracin/ml, 5 µg novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitraciri/ml, 5 µg novobiocin/ml, 1 unit polymyxin/ml. E = 5 units bacitracin/ml, 2_µg novobiocin/ml, 1 unit polymyxin/ml. F = 2 units bacitracin/ml, 2,..Mg novobiocin/ml, 1 unit polymyxin/ml. G = o units bacitraciri/ml, O JAg novobiocin/ml, O units polymyxin/ml.
= without filtration.
Talbe 15. Detailed results of plating poultry meat inoculated with strain B7988 at a level of approximately 103 organisms utilizing various combinations of selective media and methods.
SAMPLE PIATE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F' G'
16 A NIC ISO ISO NGR ISO NIC NIC NIC NIC OGR OGR B NGR ISO NGR NGR NIC NGR NGR OGR NIC OGR OGR c ISO NGR NIC NIC NIC ISO ISO NIC NIC NIC OGR
17 A NGR NGR NIC NGR NGR NIC NGR NIC NIC NIC OGR B ISO NIC ISO NIC ISO ISO NGR OGR NIC NIC NIC c NGR NGR NGR ISO ISO NIC ISO ISO NIC ISO OGR
18 A NGR ISO ISO NGR NIC NGR NIC NIC NIC NIC OGR B NGR NGR NIC NIC ISO NGR NGR ISO NIC OGR NIC c NGR NGR NIC NGR NGR ISO NIC ISO NIC NIC OGR
19 A NGR NGR NGR ISO NIC NIC NGR ISO ISO OGR OGR B NGR NGR ISO NIC NIC NGR ISO ISO NIC NIC OGR c NGR ISO NIC NIC NIC ISO NGR ISO NIC NIC OGR
20 A ISO ISO NIC NGR NIC NGR ISO ISO NIC NIC OGR B NGR NGR NGR ISO NIC NGR NGR ISO NIC NIC OGR c ISO ISO NGR ISO NGR NGR OGR ISO NIC NIC NIC ~~ ~ -- --
OGR = completely overgrown plate, ISO = isolated£• fetus, NIC = no isolated£• fetus, but isolated contaminant colonies present, NGR = no colonies present Tno growth). A = 15 units bacitracin/m1 7 15}.lg novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5.;«g novobiocin/ml, 5 units polymyxin/ml. C = 15 units bacitracin/ml, 5,,,ug novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitracin/ml, 5J-fg novobiocin/ml, 1 unit polymyxin/ml. E = 5 units bacitracin/ml, 2.}.fg novobiocin/ml, 1 unit polymyxin/ml. F = 2 units bacitracin/ml, 2 pg novobiocin/ml, 1 unit polymyxin/ml. G = 0 units bacitracin/ml, 0.-Mg novobiocin/ml, 0 units polym;Yxin/ml. I = without filtration.
a-0
Table 16. Detailed results 6of plating poultry meat inoculated with strain B7988 at a level of approximately 10 organisms utilizing various combinations of selective media and methods. - ~ -..-::::. --
SAMPLE PLATE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F' G' --- ~. =- -
21 A ISO ISO NIC NGR NGR NGR. NGR NIC NIC OGR OGR B ISO NGR ISO NGR NIC ISO NIC NIC NIC NIC OGR c ISO NGR NIC ISO NIC ISO OGR ISO NIC OGR OGR
22 A NGR. NGR ISO NIC ISO ISO NGR OGR NIC NIC OGR B NGR. NGR. NGR. NIC ISO ISO OGR NIC NIC NIC OGR c ISO NGR NGR. NIC NGR NGR. NIC ISO NIC OGR NIC
23 A NGR NGR. NIC ISO NIC NGR NIC NGR NIC NIC NIC B ISO ISO ISO NGR ISO ISO NIC ISO NIC OGR OGR c ISO NGR NGR. ISO NIC ISO ISO NIC NIC NIC OGR
24 A ISO NGR NGR. NIC ISO NIC NGR NIC NIC NIC OGR CJ'. I-"
B NGR. ISO NGR NIC ISO ISO NIC ISO NIC OGR NIC c NGR NGR NGR. NGR NIC ISO ISO ISO NIC NIC NIC
25 A NGR NGR NIC NIC ISO NGR NGR NIC NIC NIC OGR B NGR. ISO ISO NGR NGR. NGR. OGR OGR NIC OGR OGR c NGR NGR. ISO NIC NIC NGR OGR NGR. ISO ISO OGR ~~~~ ·-=- rn ~ ~
OGR = completely overgrown plate, ISO = isoltaed £• fetus, NIC = no isolated£• ~' but isolated contaminant colonies present, NGR = no colonies preserrr-C"no growth). A = 15 units bacitracin/ml, 15).'g novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5,,.ug novobiocin/ml, 5 units polymyxirl/ml. C = 15 units bacitracin/ml, 5 J-lg novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitracin/ml, 5..Mg novobiocin/ml, 1 unit polymyxi%ml. E = 5 units bacitracin/ml, 2....ug novobiocin/ml, 1 unit polymyxin ml. F = 2 units bacitracin/ml, 2;-<g novobiocin/ml, 1 unit polymyxin/ml. G = O units bacitracin/ml, O µg novobiocin/ml, O units polymyxin/ml.
= without filtration.
Table 17. Detailed results of plating poultry meat inoculated with strain B7988 at a level of approximately 109 organisms utilizing various combinations of selective media and methods. -
SAMPLE PLATE ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING
A B c D E F G A' B' F' G'
26 A NGR ISO NIC NGR NIC NGR NGR OGR NIC NIC OGR B NGR NGR NIC ISO NIC ISO OGR NIC NIC OGR OGR c NGR NGR NIC ISO ISO ISO NGR ISO NIC NIC NIC
27 A ISO ISO NGR NIC ISO NIC NIC ISO NIC OGR NIC B ISO ISO NGR ISO NGR ISO NIC NIC NIC NIC OGR c NGR NGR NGR NGR ISO ISO NGR NIC NIC OGR OGR
28 A ISO ISO NGR ISO NGR NGR NGR ISO NGR NIC OGR B NIC NIC NGR ISO ISO NGR OGR NIC NIC OGR NIC c OGR ISO ISO ISO NIC ISO OGR ISO NIC NIC OGR
°' 29 A NIC NGR NGR ISO ISO NGR NGR NIC NIC NIC OGR !\)
B NGR ISO NIC NGR ISO NIC NGR ISO NIC OGR OGR c NGR NGR NIC NGR ISO NGR NGR OGR ISO NIC NIC
30 A ISO NGR ISO ISO NIC NIC NIC ISO NIC NIC OGR B NGR NGR NIC NIC NIC ISO NGR ISO NIC OGR OGR c NGR NGR ISO NGR NGR NIC NIC NIC NGR OGR OGR -- G _ ..... ~ ... ..__ m --~
::as=;,_ -OGR = completely overgrown plate, ISO = isolated Q. f~~u~, NIC ~ no isolated £• fetus, but isolated contaminant colonies present, NGR = no colonies present (no growth). A = 15 units bacitracin/ml, 15,.v<g novobiocin/ml, 5 units polymyxin/ml. B = 15 units bacitracin/ml, 5_µg novobiocin/ml, 5 units polymyxin/ml. C = 15 units bacitracin/ml, 5µg novobiocin/ml, 1 unit polymyxin/ml. D = 5 units bacitracill/ml, 5_,ug novobiocin/ml, 1 unit polymyxin/ml. E = 5 units bacitracin/ml, 2;Ag novobiocin/ml, 1 unit polymyxin/ml. F = 2 units bacitracin/ml, 2.f-"g novobiocin/ml, 1 unit polymyxin/ml. G = O units bacitracin/ml, 0)-lg novobiocin/ml, O units polymyxin/ml.
= without filtration.
The vita has been removed from the scanned document
CAMPYLOBACTER FETUS SUBSPECIES JEJUNI FROM SOME
COMMERCIALLY PROCESSED POULTRY PRODUCTS
by
MERTON V. SMITH II
(ABSTRACT)
A method for the recovery of CampYlobacter fetus from poultry
meat using the selective methods of Millipore membrane (0.65jAm)
filtration and antibiotic (bacitracin, novobiocin and polymyxin)
medium was described and utilized to isolate the organism from the
surface of chicken necks contaminated in vitro and from retailed
commercially processed poultry products.
In a survival study one £• fetus strain remained viable for 0 5 days on the surface of poultry meat at 3 C and for 20 days at
0 -23.5 C. Two strains survived on the meat surface for 10 days at 0 -23.5 c. Three £• fetus ~· jejuni isolates were obtained from the
surface of 2 chicken necks and 1 whole dressed broiler purchased
from local retail markets. These isolates were morphologically
and biochemically indistinguishable from other£• fetus ss. jejuni
strains isolated from human and avian disease.
A discussion of the possible epidemiological implications of