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

TABLE OF CONTENTS • • • • • • • • • • e • • • • • • • • • • • • • • •• • • • • • • • • • • • • • • • •• • • • • • iii

LIST OF TABLES•••••••••••••••••••••••••••••••••••••••••••••••••••• v

LIST OF FIGURES •••• ·• .................................... 0 ••••• ••••• vii

INTRODUCTION • • • .• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1

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 •••••••••••••• ~

Vibrio fetus Classification ••••••••••••••••••••••••••••••••••

13 15

~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

iii

TABLE OF CONTENTS (Cont.)

page

SUMMARY ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 45

REFERENCES •••••••••••••••••••••••••••••••••••••••••••••••••••••••• 47

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• APPENDIX

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-

ally transmitted disease (Dalling, 1952); whereas, sporadic vibrionic

abortion of cattle and vibrionic abortion of sheep are transmitted orally

(Akkermans et~., 1956; Bryans and Shephard, 1969).

7

In addition to avian species, y. fetus has been associated with

antelopes (Trueblood and Post, 1959), goats (Dobbs and Mcintyre, 1951),

buffaloes (Mirnesiri, 1965), horses, dogs, cats (Winkenwerder, 1966),

monkeys (Valerio ~ alo, 1969), pigs (Russell, 1955), sheep and cattle

(McFaydean and Stockman, 1909; Smith, 1918; Dalling, 1952; Florent,

1959; Miller et .21.•, 1959; Lederle, 1963; Smibert, 1964a; Winkenwerder

and Maciak, 1965; Clark~ .21.•t 1969; Robards et al., 1969; Bryner~

al., 1972). Clark~ al.,(1969) reported on the incidence of y. fetus

(intestinalis) in the feces of some Australian cattle. They isolated

the organism from 79'/o of 220 heifers. They concluded that there is a

widespread incidence which is greater than these isolation findings

based on their serum antibody survey. Florent (1959) isolated y. fetus

(intestinalis) from the feces of 67% of normal calves and 7.5% of normal

cows. Lederle (1963) detected the organism from 4.3% of the samples of

calf intestinal contents. Vibrios were found in the digestive tract

of 18.5% of the cows and bulls tested by Winkenwerder and Maciak (1965).

Russell (1955) considered V. fetus to be the infective agent in certain

cases of scouring and mortality in pigs. Florent (1959) concluded that

isolates from cattle and pigs were quite similar indicating that they

may be the same organism. Smibert (1964a) isolated the organism from

24.~ of sheep between 14 weeks to 20 months old and from 4.Z{o of sheep

2 to 10 years of age. He found that these isolates greatly resembled

others from man, birds, cattle and swine (Smibert, 1964a).

Several investigators have studied the mode of transmission of

vibrio infection in cattle and sheep. Firehammer (1965) isolated

y. fetus from the feces of sheep which had been inoculated with the

organism 25 days previous. Dennis (1961) has presented a summary of

information relating to vibrionic abortion in sheep, while Robards ~

!!• (1969) reported on an outbreak of vibriosis in sheep in New South

Wales. Dennis (1967) suggested that the carrier agent in such outbreaks

is the crow, finding 65% of the birds in the area positive for V. fetus

by rectal smear. He has demonstrated that the crow can remain infected

by y. fetus for at least 42 days. Bryner ~ .s!:1.• (1972) isolated Vibrio

species from the gall bladder of 17% of the cattle and 5gfo of the sheep

they sampled in Iowa abattoirs. Seventy-four percent of the cattle

vibrios and 5gfo of the sheep vibrios were classified as y. fetus. These

isolates were capable of aborting pregnant test animals upon intravenous

inoculation. The bovine strains were not as virulent for sheep as the

sheep isolates. Winkenwerder and Bisping (1964) administered y. fetus

strains isolated from man to pregnant sheep by intravenous and oral

routes. The intravenously inoculated strain was effective in causing

the animals to abort; however, no y. fetus organisms were cultured from

the aborted fetuses. Bryans and Shephard (1969) clearly demonstrated

that V. fetus infection in sheep is capable of being transmitted by pen

contact with fecal shedders. Miller ~ ~· (1959) found that 4f:f/o of

ewes administered tissues per £§. from aborted lambs infected with

Y~ fetus and 65% of ewes given the organism by pure culture acquired a

V. fetus bacteremia. Bryner~~· (1964) were able to infect the gall

bladder of cattle experimentally inoculated by the oral route with

y. fetus (intestinalis). Infection was not achieved by oral administration

of y. fetus type 1 (venerealis). A predilection for infection in the

gall bladder by type 2 strains of V. fetus was demonstrated by Bryner

9

~.el• (1971) with various test animals. Lindenstruth and Ward (1948)

investigated possible routes of infection of ovine vibrionic abortion.

They studied the viability of y. fetus at 37°c, 20°c and 6°c in hay,

soil and manure and found that this organism can survive up to 20 days

at 6°c in all three types of suspensions. They were able to recover

the viable organisms at the three temperatures in all suspensions

after 10 days duration.

SOURCES OF VIBRIO FEI'US IN AVIAN SPECIES

Vibrios have been isolated from the liver, bile, ovary, spleen,

kidneys, heart and pericardial fluid of domestic and wild birds

(Delaplane et.al., 1955; Winterfield and Sevoian 1957; Hofstad~ ,el•,

1958; Peckham, 1958; Hagan, 1964; Truscott and Morin, 1964; Winkenwerder

and Maciak, 1964). Several studies have established the presence of

y. fetus in the ceca, intestinal contents and feces of diseased and

normal birds (Kolbl, 1964; Levina, 1964; Truscott and Stockdale, 1966;

Kolbl and Willinger, 1967; Smibert, 1969). Barnes and Impey (1970)

described a number of cecal isolates from chicken and turkey as being

uncharacterized, Gram-negative, curved rods. These bacteria resembled

spirilla morphologically and were extremely difficult to isolate and

purify. Hagan (1964) reported that 2of, of the chickens with a pre-

sumptive diagnosis of hepatitis based on the clinical pathology, flock

signs, and flock history resulted in vibrio isolations from the bile.

She isolated the organism from 0.8fo of those chickens with diseased

conditions other than hepatitis. Hofstad et al. (1958) observed what

was believed to be vibrionic hepatitis in chickens in Iowa. He cultured

10

the organism by suspending liver tissue from the infected birds in the

yolk sacs of chick embryos. Truscott and Morin (1964) have isolated

the organism from turkey poults with transmissible enteritis. Peckham

(1958) concluded that the vibrios isolated from bile 'and liver of

diseased foul in his studies were similar, if not identical, to those

organisms from other reports of avian hepatitis (Delaplane et al., 1955; . --

Winterfield and Sevoian, 1957; Moore, 1958; Moore and Grumbles, 1958;

Sevoian ~ ~., 1958). Smibert (1969) isolated y. fetus from the intes-

tinal contents of several species of wild and domestic foul. These

vibrios were similar biochemically and morphologically to isolates from

sheep and the so-called "related vibrios" of human infection. It was

suggested that these vibrios may be part of the normal intestinal flora

of birds. Winkenwerder and Maciak (1964) reported vibrio isolations

from 8&fc, of chicken from known disease infected flocks and from 36.4%

of commercially slaughtered chicken. They suggested that chicken

vibrios exist in the intestinal tract of normal birds as saprophytes

which become pathogenic under certain adverse physical conditions.

The mode of transmission of the infection in birds appears to be

identical to that of ovine vibrionic abortion and sporadic abortion in

cattle. Truscott and Stockdale (1966) considered avian vibrionic

hepatitis to be transmitted by fecal contamination. Their results

indicated that bile and cecal vibrio isolates from any one outbreak

have similar antigenic, virulent and biochemical properties. Some

. organisms were shown to be more virulent than others. Voute and

Grimbergen {1959) concluded that vibrionic hepatitis is spread by

fecal contamination within and among flocks. Waldhalm et al. (1964)

11

induced abortion in sheep by contaminating their hay with feces from

magpies experimentally infected with vibrios. Hofstad ~ al. (1972)

felt that once the infection has appeared, dissemination to other

members of the flock by fecal contamination is inevitable.

MICROFLORA OF PROCESSED POULTRY MEAT

Kraft (1971) has presented a compilation of 24 different genera

of organisms isolated from commercially processed poultry. These in-

elude such potential pathogens as Clostridium perfringens, Staphlococcus

aureus and Salmonella species. Ayres ~al. (1950) and Drewniak~ §1 ..

(1954) concluded that the organisms present on the surface of poultry

are there largely as a result of bacterial distribution from such

sources as feet, feathers, feces and intestinal contents by the pro-

cesses of evisceration, washing and handling of the birds during

commercial production.

Lillard (1971) found 5 chicken neck skins to be positive for

c. perfringens out of a total of 13 sampled. Total bacterial counts

of neck skins exhibited arithmetic means of 8.9 X 104 and 2.2 X 105

organisms/cm2 for the two processing plants investigated. Lillard

felt that the use of chicken necks in further processed products should

be scrutinized carefully due to the relatively frequent isolation of

C. perfringens from this area of the bird. Patterson (1972) found that

the neck skin of chicken frequently had a higher total bacterial count

than other areas of the bird.

Total aerobic surface counts can undoubtedly be used as an in-

dication of sanitary processing practices in the production of poultry

12

products; however, there is considerable variation in the results of

many investigators. Gunderson _tl ~· (1954) saw an increase in total

bacterial count during the processing of chickens from approximately

4.B X 1a3 organisms/cm2 on the skin of freshly killed birds to a final

count of 6.0 X 104 organisms/cm2 on the processed chilled carcasses.

Walker and Ayres (1956) also observed an increase in the counts during

commercial processing. They reported a median bacterial count of

1.5 X lcY organisms/cm2 on live birds with approximately 3.4 X 104

organisms/cm2 on the final processed carcass. Wilkerson et al. (1961) . --

in contrast, reported a decrease in total aerobes, enterococci and coli-

forms during the processing of turkey. Total aerobes declined from

about 1.0 X 106 organisms/cm2 on the live bird to 5.0 X lcY organisms/

cm2 after the final rinsing. Another report has shown a reduction in

total bacterial counts during processing (Drewniak _tl ~., 1954).

Several factors must be considered when interpreting these micro-

bial counts: 1. In some cases only one processing plant was sampled

(Gunderson~~., 1954); 2. Weather conditions can result in changes

in the bacterial load on the bird with higher levels during rainy or

snowy periods than during the drier season (Kraft, 1971); 3. Differences

in processing procedures among plants result in variations of the total

aerobic counts (Bryari et~., 1968); 4. Methods utilized for determining

total counts can influence such results.

Patterson (19?2) found that the swab-rinse method did not result

in as good a recovery as a method whereby the skin was removed and

shaken in a diluent incorporating an abrasive material. Patterson (1972)

considered the swab-rinse method "quite useful" especially in cases

13

where damage to the carcass must be avoided. Patterson (1971) reported

that swabbing the skin areas with two or three separate swabs and aver-

aging the counts resulted in a more accurate recovery of surface organisms.

The possible shelf life of refrigerated and frozen poultry meat is

greatly affected by the initial contamination after processing (Ayres

et .e,1., 1950). Cut-up chickens were found by Ayres et al. (1950) to

develop odor when a surface bacterial count of approximately 1.0 X 108

organisms/cm2 or more is reached during refrigerated storage. Slime

development occurred at just under 1.0 X 109 organisms/cm2• McVicker

et .el· (1957) considered spoilage odor to develop when the bacterial

population reaches 1.0 X 106/sq. in. Essary et .el• (1958) found that

what they considered to be spoiled chicken conta.iled just under 1.0 X 107

organisms/cm2 at the time slime formed on the surface and slightly more

than 1.0 X 106 organisms/cm2 when spoilage odor developed. May (1962)

reported a count of 1.0 X 109 organisms/gram on poultry meat that was

determined to have spoilage odor by panel members.

The organisms present on the surface of refrigerated chicken

consisted ~inly of members variously classified as psychrophilic

Pseudomonas, Achromobacter or Alcaligenes species (Ayres~ .e,1•, 1950).

Schmidt-Lorenz (1969) investigated the microflora of frozen poultry

that had been stored for 2 weeks at -30°C and found it to be made up

of 3afo Gram-positive species (corynebacteria, brevibacteria and micro-

bacteria, lactobacilli, micrococci, Gaffkya spp.) and 701/o Gram-negative

species (pseudomonads, aeromonads, Vibrio spp.) and a few yeasts.

CULTURAL AND. ISOLA.TIONMETHODS FOR VIBRIO FETUS

Kiggins and Plastridge (1956) found that V. fetus of bovine origin

14

grew best in an atmosphere of 5% o2 and 10% co2• Catalase positive

strains pr9duced an increased amount of catalase when grown in this

atmosphere; however, catalase negative strains did not become positive

under these gaseous conditions.

Plastridge and Koths (1961) found the minimum inhibiting levels

of bacitracin, novobiocin and polymyxin B sulfate for Y.• fetus to be

40 units/ml, 16)Ag/ml and 5 units/ml respectively. They reported that

the type of medium (thiol or blood agar) had a significant affect on

the inhibiting levels of bacitracin. Bacitracin had a lower level of

inhibition in thiol agar than in blood agar.

Plastridge §! 21· (1961) then investigated the efficacy of various

selective antibiotic media for the recovery of Y.· fetus from bull semen.

Bacitracin (2 units/ml) and novobiocin (2.)Ag/ml) in combination with

blood agar was superior to plain blood agar in isolating and culturing

v. fetus from bull semen. Shepler et al. (1963) reported that a com-

bination of selective antibiotic media (bacitracin-15 units/ml,

polymyxin--1 unit/ml and novobiocin-5 }Ag/ml) and Millipore filtration

(0.65,.um pore size) was superior to other methods investigated for the

isolation of Y.· fetus from bovine preputial fluid. They found that

fewer Y.· fetus colonies grew on the filtration plates. Smibert (1964a;

1972) recommended the use of both filtration and selective BN agar

(2 units bacitracin/ml and 2µg novobiocin/ml in Albimi brucella agar)

to isolate Campylobacter from fecal samples.

· Filtration of specimens through Millipore membrane filters

( O. 65 _µm pore size) was considered by Reiland and Hurvell ( 1970) to

be a valuable tool in diagnostic work with Vibrio coli. They found

15

that the vibrios passed through the filter in reduced numbers with

approximately a 2 log loss of organisms. Proteus vu.lgaris, Escherichia

.£21! and Pseudomonas aeruginosa contaminants were completely eliminated

by filtration.

Smibert (1962) developed a chemically defined medium for 87

V. fetus strains. Purines and pyrimidines were not needed by these

strains; however, nicotinic acid and one to several amino acids were

required. Utilizing this defined medium, Kowalak (1964) found that

growth of y. fetus was stimulated by lactate, citrate and succinate.

Incorporation of certain growth promoting substrances and various con-

taminant inhibitors allowed Bokkenheuser (1968) to recovery. fetus

from a mixed culture with a ratio of 360,000 aerobic bacteria to one

v. fetus.

VIBRIO FETUS CLA.SSIFICATION

Sebald and Veron (1963) suggested that the species V. fetus and

Vibrio bubulus be removed from the genus Vibrio and reclassified as a

new genus CampYlobacter based on certain distinctive characteristics

among which are their nonfermentative nature and a lower GiC content

of the DNA [30-34 moles percent (Tm)].

Various biochemical tests and serologic typing systems were

utilized by Berg et al. (1971) to revise the current v. fetus class-- - -ification. He s~ggested five V. fetus groups as follows: Serotype A

biotype 1, venereally transmitted, bovine abortion; Serotype A biosub-

type 1, venereally transmitted, bovine abortion; Serotype A biotype 2,

intestinal form, ovine abortion; Serotype B, intestinal form, ovine

abortion, sporadic abortion in cattle; Serotype c, intestinal form,

ovine abortion, avian hepatitis, human "related vibrios".

Firehammer and Berg (1965) felt that the use of the temperature

tolerance test to identify "Y.• fetus isolates is of value with limita-

tions. The same applied to the tests for tolerance to glycine, sodium

chloride and for production of hydrogen sulfide. The authors found

that not every strain that they examined could be differentiated by

these tests.

Smibert (in press) suggested several characteristics for differ-

entiating the genus Camp~obacter as shown in Table 1. Under this

classification the Q• fetus ~· fetus is the vene~eally transmitted

etiologic agent of infectious abortion or infertility in cattle.

Q• fetus~· intestinalis is orally transmitted and causes sporadic

abortion in cattle, ovine abortion and human infection. C. fetus ss.

jejuni are the so-called "related vibrios" of human infection, the

infectious agents associated with some ovine abortion and the cause of

infectious avian hepatitis. They are orally transmitted and are in-

testinal types. . Q. _ sputorum are probably nonpathogenic and reside in

the human gingival crevice and the reproductive tract of cattle, sheep

and other animals._

The classification and nomenclature used throughout the remainder

of this manuscript will be that of Smibert (in press).

la. c. b. c. c. c.

2a. c. b. c.

3. c.

17

Table 1. Some differential characteristics of species of the * genus CampYlobacter

§ s:: Q) 0 .+)

·rl •rl ro .+) .+) .+) () () Q) ::l .§ H ()

"O ro Cl) Q) Q) (/) p. Q) H H "O •rl .~ .-!

Q) E-t ro H 0 Cl) Q) Q) Q) .+) () ro ro .+) .+) s:: r-i Cl) ~:;::.; .-! ro ·rl 0 ro H H .. bO ~ 0

.+) .+) .+) (/) (/) ~o ro •rl ·rl C\l C\l ~ • ~ () s:: s:: ::r:: ::r:: <-l (<"'\ C\l

fetus subsp. fetus + + + fetus subsp. intestinalis + + -+ + + fetus subsp. jejuni + + + + -s12utorum subsp. s;eutorum + + + + + - + s;eutorurn subsp. bubulus + + + + + + d f ecalis + + ? + + + d

+ = most (9ofo or more) strains positive for this characteristic. = most (9ofo or more) strains negative.

d = some (less than 9ofo) strains positive, some negative. ? = reaction not known.

* Smibert (in press)

MATERIALS AND METHODS

A STUDY OF THE RECOVERY OF CAMPYLOBACTER FETUS FROM THE SURFACE OF POULTRY MEAT

Poultry meat for the initial recovery study consisted of a single

lot of 30 chicken necks individually packaged in sterile ''Whirl Pak"

polyethylene bags and frozen at -20°C immediately after sectioning at

a local commercial poultry processing plant (Rockingham Poultry Market-

ing Co-op., Inc., Broadway, Virginia). The necks were transported

frozen and held at -23.5°c until used in this experiment. All samples

were used within 15 days.

Each sample was allowed to thaw at room temperature for 40 minutes.

They were then aseptically cut on a sagittal plane providing two sub-

samples--one to be inoculated with a pure culture of C. fetus and the

other to be used as a control. The controls were first analyzed for

total aerobic bacterial surface count by a standard swab-rinse method

(Sharf, 1966). These counts were the average of 3 separate swabbings 2 of 2 cm each and plated in' duplicate.

_Following total surface count each control was analyzed for possi-

ble c. fetus contamination by the following procedure: The control sub-

sample was placed in an 18 ounce sterile polyethylene bag. Fifty milli-

liters of nutrient broth were added to the contents of the bag which

was then shaken for approximately 30 seconds to wash surface microorga-

nisms free. The nutrient broth surface wash was swabbed with sterile

cotton-tipped applicators onto the surface of two plain brucella agar

plates (Albimi Laboratories, Pfizer, Inc., New York) and two brucella

agar plates combining 2 units bacitracin/ml and 2,µg novobiocin/ml

18

19

which will be termed BN agar (both antibiotics were supplied free of

cost by the Upjohn Co., Kalamazoo, Mich.). The remainder of the nutrient

broth wash was then filtered through a 0.65j(m, 47 mm diameter membrane

filter (Millipore Corp., Bedford, Mass.) until a 4-5 ml portion was ob-

tained. ,The filtration process and apparatus is further described by

Smibert (1964a). The filtrate was surface plated on two plain brucella

agar plates. Bacitracin-novobiocin (BN) agar plates were swabbed with

the remaining filtrate until it was exhausted. The typical colonial

morphology of £• fetus after 4 days incubation in a microaerophilic atmo-

sphere (5% o2 , l()J& C02 , 85% N2) on BN agar is -!:,he following: colonies

of approximately 2 wm or less in diameter; smooth or mucoid; opaque or

translucent of white, cream, pink, gray or tan color; flat, thin or

convex; with round, rough or cut-glass margins. Care was taken to exam-

ine every colony present by transmitted and reflected light. Suspect

colonies were screened by growing on brucella semi-solid (0.1~ agar)

broth (referred to as semi-solid base broth) for 3-5 days. These colonies

were transferred from solid agar plates by cutting the colonies from

the agar with a sterile knife. Semi-solid broth cultures were checked

by phase contrast microscopy for shape and motility, Gram stained and

examined under bright field for typical organisms and pure culture. A

loopful of near-surface growth was then transferred to one tube of semi-

solid base broth containing 1% glucose and 0.002% phenol red and to two

brucella agar plates. One plate was incubated under microaerophilic

atmosphere.for 3-5 days. The microaerophilic plate was tested for oxidase

production, and the broth culture was examined for glucose fermentation

and catalase production by the methods outlined later in the BIOCHEMICAL

20

CHARACTERIZATION section. Cultures which could not be eliminated by

these biochemical and morphological tests were further checked with phase

contrast microscopy at different stages of growth for curved S-shaped,

gull-shaped or spiral rods and examined biochemically as shown in the

BIOCHEMICAL CHARACTERIZATION procedures.

The inoculated half of the chicken neck samples (termed the ino-

culated subsamples) were prepared by suspending approximately 109

organisms/ml, 106 organisms/ml and 103 organisms/ml in sterile 0.1%

peptone dilution buffer. One milliliter of suspension was applied to

each subsample at each level of inoculum by using a sterilized cotton

swab and spreading the inoculum over the entire surface of the sub-

sample. Five separate samples were used for each type of organism at

each level of inoculum. Strains 7480 and B7988 (Table 2) were used as

the inocula at all three levels. A total viable count of the inoculum

suspension was taken by diluting in peptone buf~er, surface plating on

brucella agar, incubating in the microaerophilic environment for 4 days

at 37°c and enumerating the colonies.

After inoculation of the neck surface, the subsamples were

immediately analyzed for .£• fetus organisms by the same procedures as

outlined previously. However, in this s.ection of the experiment the

filtered nutrient broth obtained after washing the inoculated neck

surface was swabbed onto the surface of 3 brucella agar plates with

each of the following combinations of antibiotics:

A. 15 units bacitracin/ml, 15 )Ji, novobiocin/ml, 5 units polYieyXin

B sulfate/ml (supplied free of cost by Burroughs Wellcome Co., Research

Triangle, N. C.).

Table 2. Strains of Campylobacter fetus used and their respective sources of isolation

STRAIN NO.

1-1 1261 653 B6455

B7988 B8615 C14 29A K14C T18A 3865 7480 H718 H550 H325 H840 H484 H563 · B7619

SOURCE OF ISOLATION

pigeon intestinal contents turkey (necropsy) ovine intestinal contents human female, blood and

spinal fluid human male, spinal fluid human female, blood retailed poultry meat retailed poultry meat retailed poultry meat chicken bile (necropsy) chicken liver (necropsy) chicken liver (necropsy) stool of diarrheic child stool of diarrheic child stool of diarrheic child diarrheic stool stool of diarrheic child stool of diarrheic child stool of diarrheic nurse

STRAIN IDENTITY REFERENCE

C. fetus~· jejuni Smibert (V.P.I. & S.U.) £• fetus ~· ~uni Truscott (Ontario Vet. College) £• fetus~· intestinalis Smibert (V.P.I. & S.U.) C. ~ ~· intestinalis Weaver (C.D.C.)

£• fetus~· intestinalis Weaver (C.D.C.) £• fetus ~· intestinalis Weaver (C.D.C.) £• fetus~· jejuni M. V. Smith (V.P.I. & S.U.) C. fetus~· jejuni M. V. Smith (V.P.I. & S.U.) £• fetus ~· jejuni £• fetus ~· jejuni £• fetus ~· kjuni £• fetus ~· jejuni Q.. .f.~us ~. kjuni

£• fetus ~· kjuni c. fetus ss. - -£• fetus ~· jejuni £• fetus ~· jejuni C. fetus ss. - -c. ~ ~· kjuni

M. v. Smith (v.P.I. & s.u.) Hagan (N.Y. State Vet. Lab) King (c.D.c.) King (C.D.C.) Dekeyser Dekeyser Dekeyser Dekeyser Dekeyser Dekeyser Dekeyser

(Nat. Inst. de Recherches (N.I.R.V.) Veterinaires)

(N.I.R.V.) (N.I.R.V.) (N.I .. R.V.) (N.I.R.V.) (N.I.R.V.)

N ....

22

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

antibiotics (bacitracin - - 5 units/ml, novobiocin .- - 2 )Ag/ml, poly-

myxin - -1 unit/ml) (level E in Tables 3 and 4) were only slightly

diminished. In fact, there was no significant difference (c(= 0.025)

in the isolation of c. fetus among the 6 combinations of antibiotics

when selective filtering was employed. Recovery on plates containing

no antibiotics (level G) was significantly less. There was no differ-

ence (CX:.= 0.025) between the recoveries when filtering was used and

when no filtering was undertaken and the nutrient broth wash was

plated out on selective media containing 15 units bacitracin/ml, 15).<g

novobiocin/ml and 5 units polynwxin/ml (level A' in Tables 3 and 4). The lower concentrations of antibiotics were used in combination

with Millipore filtration for the majority of the SURVEY OF RETAILED

POULTRY MEAT AND LIVER because of the following observations:

1. C. fetus isolates of natural contamination would be less

likely to grow well on artificial media as compared to laboratory

grown organisms contaminated in vitro.

2. The results of certain surveys (Smibert, 1964a; Smibert 1973)

of various strains of C. fetus indicated that the most sensitive ' ....

organisms were not affected by 2 units bacitracin/ml, 2µ.g novobiocin/ml

and 1 unit polymyxin/ml.

Table 4. Isolation of Campylobacter fetus from inoculated pou..1try meat samples expressed as a percentage of the total samples.

VARIA.BLES ANTIBIOTIC COMBINATIONS WITH AND WITHOUT FILTERING

A B c D E F G A' B' F' G'

Total samples 67 77 60 60 73 87 33 83 27 13 0

Strain 7480 (at all 67 80 53 53 73 93 27 80 33 13 0 inoculum levels)

Strain B7988 (at all inoculum levels)

67 73 67 67 73 80 40 87 20 13 0

1a3 inoculum (both strains) . \...V

50 80 70 60 60 80 60 80 30 20 o· 0

Strain 7480 40 80 60 60 60 80 40 80 40 40 0 Strain B7988 60 80 80 60 60 80 80 80 20 20 0

106 inoculum (both strains) 80 80 70 50 80 90 20 80 20 10 0 Strain 7480 80 80 60 60 80 100 0 80 20 0 0 Strain B7988 80 80 80 40 80 80 40 80 20 20 0

109 inoculum (both strains) 70 60 40 70 80 90 20 90 30 0 0 Strain 7480 80 40 40 40 80 100 40 80 40 0 0 Strain B7988 60 80 40 100 80 60 0 100 20 0 0

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.

TEST OR CHARACTERISTIC

ONPG* arginine dehydrolase* lysine decarboxylase* ornithine decarboxylase* citrate* H2S* urease* tryptophane deaminase* indole* acetoin* nitrate reduction* gelatin* glucose-K-mannitol* inositol* sorbitol* rhamnose* saccharose* melibiose* amygdalin* arabinose* catalase oxidase mannitol (phenol reduction) Gram stain coccus-bacillus motility 3.5% NaCl growth 1% glycine growth 1% bile salts growth

+ = positive test or growth - = negative test or growth b = bacillus shape G- = Gram negative alk. = alkaline reaction X = test not done

ro •r-f ..c: t>

•r-f i:-,

1l ~I t>

{I) f:r.1

+

+

+

+

+ +

+ +

+

+

x G-b + + + +

IDENTITY

{I) Q)

{I) s:: •r-f Q) {I)

{I) .-I ·r-f ;:::l ·r-f •r-f .-I Q) ,.0 .-I ro i5 ro ro u ·g {) Q) i:-, .-I ro

0... < Ii-I

x x

+ x + x + x + + x

+ + + +

+

+ +

x alk. G- G-b b + + + + + + + +

*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 ••••••••••••••••

STRAINS

+++++++++++++++++++

+++++++++++++++++++

++ww+++++++++++++++ +++++++++++++++++++ +++++++++++++++++++ +++++++++++++++++++ +++++++++++++++++++

wwwwwwwwwwwwwwwwwww +++++++++++++++++++ --++++-------------++w++++++++++++++++ ++----www+ww++w+w++

+

w

= = =

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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Soonattrakul, w., Anderson, B.R. and Bryner, J.H. 1971 Raw liver as a possible source of Vibrio fetus septicemia in man. Amer. J. Med. Sci. 261:245-249·

Spink, W.W. 1957· Human vibriosis caused by Vibrio fetus. J.A.M.A. 163:180-182.

Trueblood, M.S. and Post, G. 1959. Vibriosis as a factor in the repro-duc~ion of antelope (AntilocarEa Americana). J.A.M.A. 134:562-564.

Truscott, RoB. and Morin, E.W. 1964. A bacterial agent causing Blue-comb Disease in turkeys. II. Transmission and studies of the etiological agent. Avi~n Dis. 8:27-35·

Truscott, R.B. and Stockdale, P.H.G. 1966. Correlation of the identity of bile and cecal vibrios from the same field cases of avian vi-brionic hepatitis. Avian Dis. 10:67-73·

Valerio, D.A., Miller, R.W. and Innes, R.M. 1969. Infection of monkeys with Vibrio fetus. In "Macaca Mulatta Management of a Laboratory Br~ed:j.ng Coion~. Academic Press;-~York.

Vinzent, J •• Delarue, J. and Herbert, H. 1950. L'infection placentaire ~ Vibrio foetus. Ann, Med. 51:23-68.

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Waldhalm, D.G., Mason, D.R., MeinershagenW.A. and Scrivner, L.H. 1964. M~gpies as carriers of ovine Vibrio fetus. J.A.V.M.A. 144:497-500.

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

these findings was included.