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1988 Epidemiology of Campylobacter Jejuni. Kanagasundaram Yogasundram Louisiana State University and Agricultural & Mechanical College

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Epidemiology Campylobacterof jejuni

Yogasundram, Kanagasundaram, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1988

Copyright ©1989 by Yogasundram, Kanagasundaram. All rights reserved.

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

EPIDEMIOLOGY OF CAMPYLOBACTER JEJUNI

A dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Interdepartmental Program of Veterinary Medical Sciences

by Kanagasundaram Yogasundram B.V.Sc., University of Ceylon, 1969 M.S., Louisiana State University, 1985 December 1988 ACKNOWLEDGEMENTS

I wish to express my appreciation to Dr. Simon M.

Shane, my major professor, for all the guidance during the doctorate program and for his help in preparations of this dissertation. I wish to thank Drs. Harry V. Hagstad,

Martin E. Hugh-Jones, Richard E. Smith, Kenneth L. Schnorr,

Robert M. Grodner, and Judith L. Fishbein for being members of the examination committee and for their constructive criticisms.

My thanks are due to Blaine Elbourne for her help in preparation of this dissertation, and to Gay Wales for help in office matters. My thanks are also due to Kathleen

Harrington, Glenda Tate, and Jennifer Broussard for technical assistance in research, Michael Kearney for graphics, and Jim Roberts for research supplies.

I am grateful to my wife, Kamala, and children, Nalin and Nathen, for their support and patience throughout this program. TABLE OF CONTENTS

Acknowledgement ii

Table of contents iii List of tables v List of figures vi Abstract vii

CHAPTER 1:0 Introduction 1

CHAPTER 2:0 Epidemiology 8 2:1 Prevalence in the United States 13

CHAPTER 3.0 Source of infection 19 3:1 Avian species 19 3:2 Companion animals 21 3:3 Cattle 22 3:4 Swine 24 3:5 Other animals 25

CHAPTER 4.0 Food-borne infection 26 4:1 Introduction 26 4:2 Poultry as a source of infection 27 4:3 Other meat products 33 4:4 Milk 34 4:5 Water 36 CHAPTER 5.0 Other modes of transmission 39 5:1 Direct transmission 39 5:2 Perinatal transmission 42 5:3 Transmission by vectors 44

CHAPTER 6.0 Pathogen!sis in man 45 6:1 Clinical cource 45 6:2 Extra-intestinal infections 46

CHAPTER 7.0 Antigenic components 52 7:1 Surface proteins 52 7:2 Heat-stable antigen 53 7:3 Heat-labile antigen 53

CHAPTER 8.0 Mechanism of pathogenisis 56 8:1 Mucosal adherence and colonization 56 8:2 Mucosal invasion 58

iii CHAPTER 9.0 Serotyping 62

CHAPTER 10.0 Toxigenicity 68 10:1 Introduction 68 10:2 Toxins 69 10:3 Assays 72

CHAPTER 11.0 Conclusion 77

CHAPTER 12.0 Procedures 83 12:1 Prevalence study 83 12:2 Serotypinges 90 12:3 Toxigenicity testing 94

CHAPTER 13.0 Results 102 13:1 Prevalence study 102 13:2 Serotyping 117 13:3 Toxigenicity study 121

CHAPTER 14.0 Discussion 128 14:1 Prevalence in avian species 128 14:2 Prevalence in humans 130 14:3 Serotyping 132 14:4 Toxigenicity study 13 3 14:5 General considerations 135

REFERENCES 140 LIST OF TABLES

Isolation of C .jejuni from diarrheic human 3 patients US costs for campylobacteriosis, 1985 5

Isolation of enteric pathogens from feces 14 Isolation of C .jejuni by geographical regions 17

Extra-intestinal C .jejuni infections 51 Difference between the genera Vibrio and Campylobacter 65 Prevalence of C .jejuni in specific populations 79

Differences in the charateristics of catalase positive Campylobacter spp. 89

Species of birds examined 103

Isolation of C. jejuni from different orders of avian species 107

Isolation of C. jejuni from waterfowl 110

Etiological diagnosis in avian species yielding C. jejuni 111 Isolation of C. jejuni and Salmonella spp. from human diarrheic patients 114

Homologous and heterologous reactions of C . jejuni serotyping antisera 118 Serotypes of C. jejuni isolated from human patients and avian species 119

Enterotoxin and cytotoxin produced by C. jejuni isolates derived from either human patients or avian species 122 Most common serotypes of C. jejuni isolated: Three studies compared 134

v LIST OF FIGURES

1. Reported isolation rates of Campylobacter spp. by geographic regions, United States, 1983 16 2. Seasonal distribution of reported Campylobacter isolates by geographic regions, United States, 1984 18 3. Scheme to speciate and serotype Campylobacter isolates 84 4. Diagrammatic representation of the principle of the GMl-ELISA 97 5. Seasonal variation in isolation of C. jejuni from avian species 108 6. Seasonal variation in isolation of C. jejuni and Salmonella spp. from human patients 113 7. Age distribution of human patients with Campylobacteriosis 115

8. Comparison of C. jejuni serotypes isolated from human patients and avian species 120 9. Distribution of toxin producing C. jejuni isolates derived from human patients 124 10. Distribution of toxin producing C. jejuni isolates derived from avian species 125

11. Cytotoxin production by C. jejuni isolates from both avian species and human patients 126

12. Enterotoxin production by C. jejuni isolates from both avian species and human patients 127

vi EPIDEMIOLOGY OF CAMPYLOBACTER JEJUNI

Abstract

A study was conducted to determine the prevalence of

Campylobacter jejuni in various avian species and humans.

Cloacal swabs derived from birds submitted to the

Louisiana Veterinary Medical Diagnostic Laboratory for necropsy examination during the period February 1987 through July 1 988 were routinely cultured for C^_ jejuni using Butzler's selective medium. Campylobacter jejuni was isolated from 10.1% of the cases examined. The isolation rate was 25.2% in Galliformes, 12.9% in Anseriformes, 8.3% in Columbiformes, 7.7% in Falconiformes, and 0.6% in

Psittaciformes. None of the other orders of birds examined yielded C^_ j ej un i. Prevalence data demonstrated that

Galliformes are important reservoirs of jejuni infection for humans and that Psittacines do not represent any significant risk to human contacts.

Prevalence of C^_ jejuni in diarrheic patients was studied during a one-year period, from August 1987 through

July 1988. Of a total 1423 fecal samples cultured, 38 yielded C^ jejuni giving an isolation rate of 2.7%. This is comparable to the 2.6% prevalence rate obtained in a

similar study in Texas. Prevalence rate for Salmonella

spp. during the same study was 2.0%. Seasonal fluctuation

in prevalence of C_._ jejuni in humans was demonstrated, with

a peak in Summer and a smaller increase in Winter. A high

incidence was observed in children under 5 years, and in

adults aged 21 to 25 and 31 to 40 years.

The Penner serotyping system was able to classify

91.1% of the isolates obtained from human patients and

96.7% from avian species. Fourteen of the 16 serotypes

considered in the present study were identified among the

isolates. Eight of the serotypes were common to both avian

species and humans indicating the potential for zoonotic

transmission of Cj_ jejuni infection from birds. Serotype

Penner 1 was isolated only from avian species indicating

the low risk this serotype poses for humans while serotype

Penner 10 was isolated mainly from humans suggesting that

this serotype is transmitted by direct contact or acquired

from non-avian species.

Two serotypes (Penner 5 and 10) showed consistent

production of enterotoxins. Toxin was produced by 75% of human and 85% of avian isolates demonstrating the

considerable potential for human infection with toxigenic

strains of jejuni.

viii EPIDEMIOLOGY OF CAMPYLOBACTER JEJUNI

Chapter 1

INTRODUCTION

Campylobacteriosis has received considerable attention as a food-borne zoonosis during the past decade (Butzler and Skirrow, 1979; Blaser et al., 1979; Shane and Montrose, 1985). Recovery of Campylobacter jejuni from human cases of enterocolitis has stimulated investigation of the epidemiology and pathogenesis of the disease (Drake et al. , 1981; Coffin et al., 1982). The pathogen occurs frequently in the intestinal tract of food animals

(Prescott and Munroe, 1982; Garcia et al., 1983), including cattle (Prescott and Bruin-Mosch, 1981), sheep

(Bolton et al., 1982), goats (Firehammer and Myers, 1981) and chickens (Simmons and Gibbs, 1979).

The role of animals as reservoirs of campylobacteriosis has been investigated since the recognition of the disease

(King, 1957). Campylobacter jejuni infection may be transmitted by undercooked poultry (Brower et al., 1979),

1 unpasteurized milk (Blaser et al., 1979 ), undercooked red meat (Yangisawa, 1980), seafood (Blaser, 1982) and untreated surface water (Vogt et al., 1982). Prolonged survival of the organism in contaminated meat, even when stored at low temperatures (Yogasundram and Shane, 1986), increases the probability of food-borne infection.

The development of selective media for the isolation of

£' 7ejvni in the early 1980's, facilitated research and investigation of outbreaks of campylobacteriosis. Blaser

(1982) reported that C. jejuni is isolated as a primary pathogen from humans more frequently than either Salmonella spp. or Shigella spp. Many hospitals and health care facilities now routinely screen feces from diarrheic patients for Campylobacter spp.

Isolation of C. jejuni from clinical cases has been reported from most industrialized and third world countries (Table 1).

The average length of illness following C. jejuni infection is 11 to 17 days in contrast to infection with Salmonella spp. which may be 5 to 12 days in duration

(Harris et al., 1986). Hospitalization was more frequently required to treat campylobacteriosis (6.7% cases) than for Salmonellosis (3.5%). Table 1. Isolation of C. jejuni from diarrheic human patients.

Country Reported by Year Prevalence

Q. ”6

Australia Steele and McDermott 1976 5.8

Britain Bruce et al 1977 13.9 Africa Demol and Bosman 1978 11.0

France Delorme et al 1979 9.0 Indonesia Ringertz et al 1980 4.8 United States Young et al 1980 4.7

Bangladesh Stoll et al 1982 14.0

India Nair et al 1983 10.0 Brazil Fernandez et al 1985 7.4

China Young et al 1986 11.8

I reland Lafong and Bamford 1986 2.0 The relative significance of the two infections is reflected in the estimated costs associated with salmonellosis and campylobacteriosis in the United States

(Morrison and Roberts, 1986). The total annual cost

attributed to campylobacteriosis in the US during 1985 was

estimated to range from $ 720 million to $ 1.4 billion

depending on certain assumptions regarding the value of

human life. The comparable figure for salmonellosis ranged from $ 670 million to $ 1.2 billion. The derivation of costs is provided in Table 2.

An estimated 2.1 million cases of campylobacteriosis occur in the United States annually, including 2,100 fatalities (case fatality rate 1:1000). A study conducted in Seattle (1985) indicated that approximately 50% of the

cases of campylobacteriosis were directly or indirectly associated with consumption of poultry. The annual costs attributed to poultry-related campylobacteriosis range from $ 350 million to $ 700 million, justifying research into methods of control and prevention.

Reduction or elimination of C. jejuni contamination in poultry and meat is an essential step in reducing losses due to campylobacteriosis. An estimated 1.8 million cases of salmonellosis and campylobacteriosis are attributable to consumption of beef and chicken (Menning, 1988). Due Table 2. U.S. Costs for Campylobacteriosis, 1985

Costs Cases Total Estimate Cost categories per case $ in : millions $ Number Low High Medical Costs

Mild cases 0 2,042,660 0 0 or 1,931,975 Moderate cases 938 57,340 53.8 157.6 and deaths 168.025

Lost productivity

Mild cases 230* 2,042,660 469.8 444 . 4 or 1,931,975 Moderate cases 289** 55,340 16.0 48.0 or 166,025

Deaths: Human capital method 85,800 or or 2,100 180.2 738.2 Adjusted human 351,500 capital/willingness to pay

Miscellaneous*** 62 57,340 3.6 10.4 or 168,025

Total $723 to $ 1,399

* This productivity loss estimate for mild cases also includes some miscellaneous costs of undetermined value. ** This is a low estimate because it omits the value of homemaking. *** Miscellaneous costs include transportation, child care, laboratory tests, acquiring employment.

Source: Morrison and Roberts, 1986 to the high population density in commercial broiler and turkey farms, transmission of C. jejuni between and within

flocks proceeds rapidly and contributes to high rates of

prevalence (Prescott and Gellner, 1984; Annan-Prah and

Jane, 1988). Transmission by flies and coprophagy

facilitate the spread of C . jejuni infection (Shane et al., 1985). Although growing broilers on wire floors will theoretically reduce infection, this system is neither

technically nor economically feasible. Biosecurity and

surveillance will reduce, but not eliminate, infection and transmission of C. jejuni at the farm level.

Competitive inhibition as exemplified by the 'Nurmi Concept' involves the introduction of a complex of

microorganisms from the intestinal tract of mature birds

into newly hatched chicks to suppress infection with both C. jejuni and Salmonella spp. (Nurmi and Rantala, 1973).

Though this procedure has shown promise in eliminating Salmonella infection, it has been shown to be inefficiant

in eliminating C. jejuni (Shanker et al., 1988).

Implementing this procedure on a commercial basis is

impractical using existing technology (Hudson et al., 1987 ) .

Antibiotics such as tetracycline in feed suppress bacterial infection and show an increase in weight gain

for broilers. Under practical conditions, infection re-occurs in flocks during the mandatory withdrawal period for antibiotics upto 5 days before depletion.

Cross contamination between broiler carcasses occurs in the processing plant (Luechtefeld et al., 1982). Studies indicate isolation of C. jejuni in 65% of carcasses at retail outlets (Hosie et al., 1982). This suggests that chlorination added to processing plant chill tanks is not effective in eliminating C. jejuni (Cruickshank, 1982). Low dose irradiation was demonstrated to be a more effective decontaminant, than chemicals (Yogasundram et al., 1987). A radurization dose of 1 KGy effectively eliminated C. jejuni surface contamination at a level of

10 Colony Forming Units (CFU) per cm on chicken drumsticks. Subjecting poultry meat and seafood to low- dose irradiation after packing, represents an effective method of eliminating C. jejuni contamination. Chapter 2

EPIDEMIOLOGY

Campylobacter jejuni is now accepted as a major cause of enterocolitis in humans (Skirrow, 1977). The prevalence of C. jejuni infection often relates to diagnostic dilligence and the thoroughness of laboratory procedures. The microaerophilic and thermophilic growth requirements of this organism demands special media and

isolation techniques. The development of selective media, incubation at 42°C, and the use of commercial gas mixtures have contributed to an increased recovery rate of

£ m jejuni from animals and man (Butzler and Skirrow, 1979) .

The prevalence of intestinal campylobacteriosis is extensively documented and isolation from clinical cases has been recorded in many developed and third-world countries (Table 1). Recovery of C. jejuni from feces of diarrheic patients exceeds the isolation rate of

Salmonella spp. (Harris et al., 1986a)

8 Identification of C . jejuni in 8 to 13% of diarrheic

patients in developing countries has been reported by Bokkenheuser and Sutter (1981) in contrast to only 4 to 7% from cases in industrialized countries. The corresponding recovery rates for C . jejuni from clinically unaffected

individuals are 5 to 17% and 0 to 1% respectively in these two catogories.

The magnitude of the problem in developing countries is generally underestimated since the prevalence rate is a

function of of C. jejuni isolation. With the exception of

structured surveys, reports are limited in contrast to the

situation in developed countries, where routine examination for Campylobacter spp. is undertaken and where national reporting system exist (Blaser et al.,1983a). The relatively high rate of recovery from clinically

unaffected individuals in developing countries complicates

an appraisal of the significance of C . jejuni in diarrheic patients ( Bokkenheuser and Sutter, 1981).

Blaser et al. (1980a) reported an isolation rate of 18%

in children aged 1-5 years in rural Bangladesh.

Aboriginal Australian children are healthy carriers of

C. jejuni with an isolation rate of 17% according to Berry et al.(1981). Rajan and Mathan (1982) demonstrated a 15% prevalence in all ages of healthy individuals in Southern India. Lower rates were reported in Ethiopia,(3%) (Thoren et al., 1982); Gambia,(4.2%) (Billingham, 1981); Zaire, (6%) (Butzler, 1978) and Central Africa (3%)(De Mol et al., 1980).

In developed countries, isolation of C. jejuni in feces is uncommon unless patients present with diarrhea. Several surveys indicated a prevalence of only 1% in clinically normal individuals. In Belgium, isolation from

healthy individuals and diarrheic patients attained 1.3%

and 5.1% respectively (Butzler et al., 1973). In England

rates were 0.6% and 13.9% (Bruce et al., 1977); In the Netherlands 0% and 10.8% (Severin, 1978); France 0% and 9% (Delorme et al., 1979); and in The United States 1.2% and 4.7%, (Young et al.,1980). Bruce et al. (1977) recovered

C. jejuni from 39 out of 280 patients with acute

gastroenteritis in Hereford, England. In contrast, these authors recovered C. jejuni from only 2 out of 117

contacts and only 1 out of 156 non-diarrheic patients.

Drake et al. (1981), isolated C. jejuni from 63 of 1953

fecal samples of diarrheic patients in the Mayo Clinic during 1979, in contrast to an isolation rate of 2% for

Salmonella spp. and Shigella spp. combined. Goodman et al. (1981) examined 2059 fecal samples from hospital patients and food handlers. A total of 26 were positive for C. jejuni and of these 96% showed acute diarrhea. In Bangladesh, Stoll et al. (1982) recovered C. jejuni from 14% of 3550 patients included in a survey of infants and adults. Rajan and Mathan (1982), reported on the

isolation of C. jejuni from 15% of a random sample of

healthy individuals in Southern India. In contrast, Nair, Battacharya and Pal (1983) isolated C .jejuni from only 10% of the 116 acute cases of diarrhea admitted to an infectious disease hospital in Northern India. In half of

these confirmed cases, C. jejuni was recovered in conjunction with Vibrio cholera.

Seasonal fluctuation in the incidence of

campylobacteriosis has been reported by many investigators. An epidemiological survey conducted in Texas on C. jejuni (Subramanyam et al., 1984) showed an

isolation rate of 2.3% (41/1840) from the feces of patients with diarrhea. An increase in positive

isolations (40%) occured during spring. Higher isolation

rates in the United States during summer months was reported by Goodman et al. (1981). Similarly, an increase in prevalence during summer was documented in Belgium

(Lauwers, Deboeck and Butzler, 1978), in Switzerland

(Graf, Schar and Heinzer, 1980) and in the Peoples

Republic of China (Tang et al., 1984). Studies have been conducted to analyse the age distribution of C. jejuni infection in humans. In an epidemiological survey in Bangladesh, Glass et al. (1983) reported the highest rates of C. jejuni recovery in healthy infants. An isolation rate of 32% was reported in children less than one year old while an overall isolation rate of 19% was attained by children under five years.

Prevalence in diarrheic children less than 12 months old and under 5 years of age was 25% and 20% respectively. In contrast, the prevalence in diarrheic patients over 5 years old was only 6% and in healthy individuals 3%.

In a study involving 435 patients with campylobacter enteritis in Sweden, Walder (1982) demonstrated two peaks in age distribution. The first included infants up to 5 years old and the second comprised individuals aged 20-35 years.

Abbott et al. (1984), documented an increase in

C. jejuni infection in infants proportional to age. No isolates were obtained from infants under 1 month of age.

An isolation rate of 3% occured in the 1-12 month age group, 6% from 12-24 months, and 7% in children 2-5 years of age. Tang et al. (1984) reported that children from

10 days to 8 years of age were most frequently affected in the Peoples Republic of China. A seroepidemiologic study conducted in England by Jones and Robinson (1981), indicated an occupational component to C. jejuni infection. The study revealed

significant titers in 27% and 68% of agricultural workers

employed in poultry and cattle abbattoirs respectively

compared to 2 to 5% in controls engaged in crop production. Blaser et al. (1979) implicated occupational exposure in three patients with C . jejuni infection. One

was employed as a meat inspector in an abattoir, a second

was a worker in a poultry processing plant and the third

was a child of an abattoir worker.

2:1. Prevalence in the United States

A multicenter study conducted in 1980/1981 by Blaser et al. (1983a) showed a 4.6% prevalence of C. jejuni in the United States. A total of 8097 fecal specimens from 8 hospital laboratories in the United States were examined during a 15 month period. In Comparison to C. jejuni,

Salmonella spp. and Shigella spp. yielded prevalence rates of 2.3% and 1.0% respectively. Positive fecal cultures were more frequently identified when samples were obtained within a 7-day period following onset of illness.

The prevalence of C. jejuni in 8 states is depicted in Table 3. Table 3. Isolation of Enteric Pathogens From Feces

State Specimens Specimens positive for

Studied Campylobacter Salmonella Shigella Any of

the Three

n %

Michigan 636 9.3 1.7 0.5 11.5 o Maryland 573 1.4 7.9 « VO 10.2

Illinois 2947 6.7 2.4 0.3 9.4

Georgia 160 2.5 2.5 4.4 9.4

Colorado 352 6 . 0 1. 4 0.9 8.3

Oregon 797 5.6 1.8 0.8 8.2 -4 I to Oklahoma 751 • 2.0 4.3 7.5

California 1315 2.1 1.3 1.0 4.4

Source: Blaser et al., 1983. Campylobacter jejuni was isolated from 8282 patients in 42 states in the United States during the period January

to December 1983 (Riley and Finch, 1985). These authors

reported the highest frequency from infants (18/100,000

population) and the lowest rate (3/100,000) from subjects over 55 years of age. Isolation was highest during summer with 46% of the isolates during the period June through

September. Of the 9 geographical regions studied, the New

England region (Maine, Massachussettes, New York) showed

the highest isolation rate (20.8/100,000) while the West

South Central region (Texas, Oklahoma, Arkansas, Louisiana) showed the lowest isolation rate (0.3/100,000), as recorded in Figure 1 and Table 4.

Tauxe et al. (1987), in a study conducted in 1984, reported 8837 C. jejuni isolations and 8 outbreaks of campylobacteriosis. A national isolation rate of

4.9/100,000 population was recorded. As in the earlier

studies, infants showed the highest infection rates

(11/100,000) followed by adults aged 25 to 29 years. There was seasonal fluctuation, with a higher incidence during summer. In the Northeast and Southeast there was a peak commencing in June and extending through mid

September followed by a second smaller peak in November.

In the west, the summer peak occurred approximately one month earlier than the corresponding peaks in the

Northeast and Southeast regions (Figure 2). Alaska PER 100,000 POPULATION HawalI □ BEGAN REPORTING REGULARLY JAN. 1983 □ BEGAN REPORTING REGULARLY LATER IN 1983 □ DID NOT REPORT

Figure 1. Reported Isolation rates of Campylobacter species by geographic region, United States 1983, Regions: E.N.C., East North Central: E.S.C., Eas1 South Central; M.A., Mid-Atlantic; MT,, Mountain; H.E., New England; PAC., P acific; S.A., South A tlantic; W.N.C., West North Central; W.S.C., West South Central. Types of surveillance activity: L, laboratory-based only; M, mandatory; V, voluntary; N, none of these.

Source: Riley and Finch, 1985. PERCENT OF REGIONAL TOTAL 1 1 Source: iue . esnl itiuin f eotd Campylobacter reported of distribution Seasonal 2.Figure 2 • A FEB OCTNOV MAR AUG DEC SEPJAN MAY APR JUL JUN ax e a et Tauxe 1. , 1987 slts y egahcl ein Uie States, United region, geographical by isolates West 1984. ortheast N Southeast AE F REPORT OF DATE

17 Table 4. Isolation of C. jejuni by geographic region

Region Prevalence per 100,000 population

New England 20.8

Pacific 13.9

Mid-Atlantic 11.9 South Atlantic 8.6

West North Central 8.2

Mountain 4.8

East North Central 3.2

East South Central 1.8

West South Central 0.3

Source: Riley and Finch, 1985 Chapter 3

SOURCE OF INFECTION

Many animal species have been recognized as carriers of C. jejuni representing a potential source of infection for humans (Smibert 1978; Skirrow 1982).

3:1. Avian species

Commercial poultry, and to a lesser extent free living avian species, are reservoirs of C. jejuni. Simmons and Gibbs (1979) isolated the organism from 72% of a batch of broilers delivered to a processing plant. Grant et al. (1980), isolated C. jejuni from 85% of cloacal swabs derived from live broilers in a market. Svedhem and Kaijser (1981) reported a recovery rate of 36% in broiler chickens. Smitherman et al. (1984) documented an isolation rate of 15% in cloacal swabs and feces obtained from four farms in California while Prescott and Gellner

(1984) recovered C. jejuni from the colon of 28 out of 60 chicken flocks reared on Canadian farms.

19 The potential routes of entry of C. jejuni into a flock of chickens, include lateral infection of new-born chicks from older birds, indirect contact from fomites or contaminated feed and water (Blaser et al., 1983b).

Generally there are no obvious signs of illness associated with intestinal carriage of C. jejuni by chicken (Ruiz-Palacios, 1983).

Prevalence rates in domestic poultry ranging from 30% to 100% have been reported by various workers: Bruce et al. (1977) documented an isolation rate of 68% from a commercial chicken flock. Ribeiro (1978) recovered

C. jejuni from 31 out of 34 broilers. Goren and de Jong (1980) isolated C. jejuni from 30% of 239 chicken and 42% of 89 flocks of commercial broiler chickens. Grant et al.

(1980), recorded C. jejuni from 38 out of 46 rectal swabs obtained from live birds at a market. Isolation rates of up to 100% (Luechtefeld and Wang, 1980) have been obtained from fecal specimens from turkeys and 88% from sampling domestic ducks (Prescott and Bruin-Mosch 1981). C. jejuni was isolated from 35% of a small sample of free living ducks (Luechtefeld et al., 1980) and 8% of samples derived from non-domestic pigeons (Luechtefeld et al., 1981).

Harris et al. (1986b) reported isolation of C. jejuni from 57% of 297 broiler chicken carcasses at a processing plant, 23% of 862 carcasses at point of sale and 17% of 29 game-hen carcasses at a retail market. Contamination rates determined at regular intervals in the subject plant

increased as the week progressed and a higher rate of

recovery was obtained from samples collected later in the

day. Eighty five percent of chicken livers and 89% of gizzards yielded C. jejuni in a study conducted by Christopher et al. (1982).

3:2. Companion animals

Companion animals, especially dogs and cats, can transmit C. jejuni to humans (Skirrow 1977; Blaser et al.,

1978; Vandenberghe et al., 1982). Hosie, Nicholson and

Henderson (1979) identified C .jejuni in 11% of 179 canine

fecal samples in Scotland but the authors did not detect any difference in isolation rate between diarrheic and non-diarrheic dogs and concluded that C. jejuni is not a

primary pathogen in this species. Ferreira et al.,

(1979), in contrast reported a higher prevalence in diarrheic dogs (90%) compared to only 25% in clinically unaffected subjects. Blaser et al. (1978) implicated pups as a potential source of human campylobacteriosis.

Wright (1982) isolated C. jejuni from 5% (12/260) of canine fecal samples collected in a public park. Svedhem and Norkrans (1980) indicated that C. jejuni could be

isolated from domestic cats and Patton et al. (1981)

recorded a 14% prevalence in this species.

3:3. Cattle

Campylobacter jejuni is an intestinal commensal of cattle (Blaser et al.,1983b) and individual cows can

excrete the organism in feces intermittently for several months (Robinson, 1982). Herds may be infected with multiple serotypes at any one time (Taylor et al., 1982).

In a study conducted by these authors, one dairy herd

yielded Penner serotypes 4,7,16 and 37, a second, Penner serotypes l,2and 7 while a third showed serotypes 7 and

11. Bryner et al. (1972) recovered C. jejuni from 17% of

bile samples derived from slaughtered beef cattle in Iowa.

Gill and Harris (1982) suggested that unweaned calves were more likely to serve as reservoirs of C. jejuni than mature cattle. In this study, a relatively high prevalence of 45% of fecal samples from unweaned calves yielding C. jejuni was attributed to cross contamination during transport and holding before slaughter. Excretion of C. jejuni in milk has been documented under both field and experimental conditions, Lande r

(1982), inoculated 13 quarters of 7 dairy cows with C. jejuni at doses ranging from 1 to 103 CFU per quarter,

Mild to acute mastitis was induced and C .jejuni could be recovered from milk for up to 75 days after infection. During the acute phase, C. jejuni could be detected at

levels ranging from 102to 106 CFU per ml of milk. Svedhem

and Kaijser (1981) documented a 19% isolation rate for C. jejuni derived from cattle feces at an abattoir while Luechtefeld and Wang (1982) reported a prevalence of 43% in cattle cecae.

Humphrey and Beckett (1987) reported isolation of C. jejuni from 83% of 12 dai ry cattle herds, with 10% to

72% animals demonstrating coloni zation of the intestinal tract. The authors observed that few cows were positive for C. jejuni at more than one sample interval giving support to the observation of Robinson (1982) that

C. jejuni can be excreted intermittently by cattle, Affected herds in the sample yielded a prevalence rate of 8.1% in milk samples. 3:4. Swine

Swine may carry C. jejuni as an intestinal commensal (Luechtefeld and Wang, 1982). They reported 66% recovery

of C. jejuni in 71 fecal samples collected from pig farms. Stern (1981) recorded an 87% (33/38) recovery rate from feces and a 22% (13/58) from carcasses at slaughter. Similarly, in West Germany Sticht-Grah (1982) reported

that 59% of herds were infected including 70% of fecal

samples, 75% from ingesta and 45% from washed intestines. Intestinal tracts which were sampled before and after salt treatment yielded 50% and 42% respectively demonstrating

only a small decrease in contamination. Slaughter

equipment and the abattoir environment yielded 33% positive samples.

In a study of abattoirs and butcher's premises, Bolton et al. (1982), demonstrated that 56% of slaughtered pigs were intestinal carriers of C. jejuni. Svedhem and

Kaijser (1981) reported a 95% prevalence rate in pigs sampled at slaughter. 3:5. Other animals

Zoo and laboratory animals can serve as a potential source of C. jejuni infection. Luechtefeld et al. (1981) documented C. jejuni in 6% of 575 fecal specimens derived from animals in a United States zoo. The organism has been isolated from clinically normal laboratory animals including hamsters, ferrets and rats (Fox, 1982). Among primates surveyed, baboons and two species of monkeys

(Hacaca fascicularis, Erytrocibus patas) were found to be healthy carriers of C. jejuni with a prevalence rate of 17.8% (Tribe et al., 1979). Chapter 4

FOOD BORNE INFECTION

4:1. Introduction

Outbreaks of campylobacteriosis ascribed to consumption of contaminated raw or undercooked food have been extensively documented (Blaser et al., 1983a). Poultry and red meat, unpasteurized milk, shellfish and other seafood have been implicated in outbreaks of campylobacteriosis.

Poultry meat and raw milk are more frequently incriminated than other food items. This is consistent with studies which show that between 60% to 90% of packaged poultry meat are contaminated with C. jejuni (Luechtefeld and

Wang, 1981; Kinde et al., 1983). Infection is associated with a high prevalence of C. jejuni in broilers reared on litter and the extent of cross contamination at various sites in poultry processing plants.

Certified raw milk is a potential source of C. jejuni infection. Infected cows may excrete the organism in milk which may also be contaminated by feces.

26 4:2. Poultry as a source of infection

Poultry meat was initially suggested as a source of human campylobacteriosis by King (1962). Avian species

serve as a productive host of C. jejuni (Prescott and Monroe, 1982) and the organism has been isolated from the

intestinal tracts of chickens, turkeys, domestic ducks, migratory waterfowl, and free ranging pigeons. Studies have shown that commercial and backyard poultry are

frequently infected with C. jejuni (Ribeiro, 1978). It is

accepted that infection is acquired during the growing

period and neither vertical transmission nor hatchery contamination occur (Oosterom, 1985). Campylobacte r

jejuni could not be isolated from 850 samples derived from

hatcheries, suggesting infection after placement of flocks

(Oosterom and de Wilde, 1983). These workers also documented the isolation of C. jejuni from feces and litter after two to four weeks of rearing despite the fact

that the day old chicks, feed, litter and environment of houses were negative for C. jejuni at the begining of the the investigation.

Demonstration of C. jejuni in cecal contents, meat and skin of broilers (Hayek and Cruickshank, 1977) and the report by Bruce et al. (1977) concerning the similarity in biotype of chicken and human isolates has prompted investigations concerning the mode of transmission of C. jejuni from poultry to humans. Simon and Gibbs (1979) showed that C. jejuni present in the intestinal tract before slaughter could survive commercial processing.

These authors isolated C. jejuni from the cecal contents of 36 out of 50 carcasses immediately after evisceration and subsequently at the end of the processing line. After refrigeration and storage, C . jejuni could be isolated from 67% of carcasses which were sampled. Smeltzer (1981) isolated C. jejuni from 94% of fresh, commercially processed chicken carcasses.

Oosterom et al. (1983) demonstrated a high rate of introduction of C. jejuni into two broiler processing plants in the Netherlands. The organism was detected on feather plucking machinery and evisceration and packing areas, resulting in significant contamination of poultry meat. Cross infection of carcasses during processing was investigated by Wempe et al. (1983) who demonstrated the role of feather pluckers and chilling tanks in dissemination of C. jejuni. The organism was isolated from 60-100% of cecal samples of broilers in which levels of 106 CFU/gram of ingesta were attained. Luechtefeld and Wang (1981) examined fecal, rectal and cecal swabs from 600 turkeys delivered to a USDA-approved processing plant. Campylobacter jejuni was recovered from

100% of fecal samples which were examined. The organism was also isolated from conveyors in the packing area, waste gutters and water treatment lagoons. Christopher, Smith and Vanderzant (1982) reported the presence of

C. jejuni in 85% of chicken livers and 89% of gizzards sampled immediately after evisceration.

Yusufu et al. (1983) conducted a prevalence study in two California turkey processing plants. They reported isolation rates of up to 23% from feathers, 6% in scald water overflow, 95% in drip water from feather pickers,

78% in recycled water used to clean gutters, 93% from ceca and 61% from water used to wash carcasses. Defeathering equipment contributed significantly to cross infection.

Marjai et al. (1982) reported isolation of C. jejuni at every phase of chicken processing in a study conducted in Hungary. A prevalence rate of 84% was obtained from intestinal samples. Of 118 specimens derived from carcasses ready for delivery to consumers, 88 (75%) were found to be contaminated. Campylobacter jejuni has been recovered from poultry meat at retail outlets, suggesting that the organism can

be transmitted from poultry to man as a food-borne

infection. Park, Stankiewicz and Lovett (1981) examined whole chicken carcasses from 20 different food stores in Ohio and Ontario, over a 5-week period. The authors recovered C. jejuni from 54% and 62% of the carcasses

respectively. Kinde, Genigeorgis and Pappaioanou (1983) noted an 83% recovery rate in packaged chicken wings in California. The isolation rate declined to 16% after cold storage for 6 days. Rayes, Genigeorgis and Farver (1983) recovered C. jejuni from 50% to 60% of fresh and frozen turkey wings. Giblets were shown to be the most frequently contaminated turkey portions, with isolation rates attaining 42% (Dromigny et al. 1985).

The extent of C. jejuni infection in live chicken, ducks and turkeys and their products suggest that these species represent a significant zoonotic potential for consumers.

Shanker et al. (1982) demonstrated that 75% of a sample of broiler flocks in Australia were infected with

C. jejuni and that the organism was present in 45% of carcasses which were examined. A concurrent study indicated that 98% of 46 C. jejuni isolates derived from

humans with enterocolitis and 82% of 19 isolates from

broilers correspond to biotype I. This suggested an epidemi- ological relationship between the consumption of

broiler meat and enteric diseases in humans.

Contaminated chicken meat has been demonstrated to be the cause of C. jejuni infection in man. Hayek and Cruickshank (1977) reported that prepared chicken was associated with gastroenteritis in 5 out of 25 guests at a reception. All patients demonstrated an elevated agglu­ tinating antibody titre to C. jejuni and the organism was

recovered from one individual. In the Netherlands, 89 out of a group of 123 cadets developed diarrhea after consumption of undercooked chicken (Brouwer et al. 1979). Campylobacter jejuni was recovered from 33% of 85 fecal samples which were examined. Consumption of poultry meat was the cause of two out of five food-borne disease outbreaks investigated by the Center for Disease Control in the U.S. in 1980 (CDC 1983) and C. jejuni was implicated in both outbreaks involving 170 cases.

In Sweden an outbreak of campylobacter enteritis among the staff of a poultry abattoir was reported by

Christenson et al. (1983). During a holiday season inexperienced teenage workers replaced some of the regular staff. Within a week, 40% of the employees developed gastroenteritis but 71% of the substitutes were affected compared to only 29% of the regular workers.

Campylobacter jejuni was isolated from the feces of 76% of substitutes and 55% of the regular employees. Contaminated poultry carcasses are a potential source of C. jejuni infection for abattoir workers and campylobacte­ riosis may be regarded as an occupational disease, especially if improper handling and deficiencies in personal hygiene exist.

Oosterom et al. (1984) conducted a retrospective study of household contacts of patients yielding C. jejuni. An isolation rate of 8.5% was obtained compared to 5.1% for

Salmonella spp. and 1.4% for Shigella spp. The authors implicated chicken meat as a source of infection particularly when barbeque cooking was used. There was no significant association between infection and consumption of pork, beef or mutton. Similar findings were reported by Deming et al. (1987) in Georgia who employed a case control technique to identify risk factors for C. jejuni infection. When age, sex and residence were matched the odds ratio for eating undercooked or raw chicken was calculated to be 49%, compared to only 7% for eating fully cooked chicken. This study confirmed that raw,

undercooked or barbequed chicken is a highly significant risk factor for C. jejuni infection.

Rosenfield et al. (1985) reported the first outbreak in Australia in which C. jejuni was isolated from both human patient and their food. The isolates were demonstrated to

be serologically identical. A chicken casserole was identified as the source of infection for 7 out of 17 affected guests. A food specific attack rate of 58% was

calculated for the implicated chicken meal. Penner

serotype 18(21,29) was isolated from 3 of 4 symptomatic patients and also from 3 samples of raw chicken, represen­

tative of the batch used to prepare the meal.

4:3. Other meat products

Consumption of undercooked hamburger was suggested as the source of infection for an outbreak of Campylobacter enteritis in the Netherlands (Oosterom et al. 1984) while undercooked pork was regarded as the vehicle in a Japanese study (Yanagisawa 1980). Raw clams were shown to be the cause of an outbreak of campylobacteriosis in New Jersey where 18 out of 31 subjects at risk were clinically

affected (Blaser, 1982). Turnbull and Rose (1982)

reported isolation of C. jejuni from minced beef in a

retail outlet demonstrating that red meat could serve as a possible source of infection.

Strum (1986) documented a case involving a 50 year old woman who developed campylobacteriosis following consumption of raw oysters. Fecal samples, which were

subjected to examination following a 9 month history of

intermittant diarrhea, yielded heavy growth of C . jejuni.

Diarrhea ceased following administration of erythromycin.

4:4. Milk

Unpasteurized milk is a frequent source of C. jejuni infection in humans. Taylor, Weinstein and Bryner (1979) reported 3 confirmed cases of campylobacteriosis,

following consumption of certified raw milk in Los

Angeles. Blaser et al. (1979) reported on an outbreak of gastroenteritis in which 3 members of a family became ill after consuming milk from a cow which subsequently yielded

C. jejuni. Porter and Reid (1980) recorded a case of

Campylobacter enteritis with an attack rate of 50% 35 following consumption of unpasteurized milk. The organism was isolated from both sympt CIuCi tl C and asymptomatic consumers and from milking equipment.

Jones et al. (1981) documented an extensive outbreak in which 2500 children were infected after consuming milk contaminated with C. jejuni. Thirteen outbreaks of campy­ lobacteriosis associated with raw milk were reported in England and Wales between 1978 and 1981 (Robinson and

Jones, 1981). Potter et al. (1983) documented an outbreak in Georgia involving 50 patients in 30 households who developed gastroenteritis attributed to C. jejuni. Milk from a certified dairy was the common source of infection and fecal samples from cows in the herd yielded C. jejuni.

Commercial cake icing was responsible for an outbreak of campylobacteriosis in Connecticut (Blaser et al., 1982).

Considering the fact that a large number of milk-related outbreaks of campylobacteriosis have been recorded, the possibility of cattle being an important reservoir of the organism cannot be ignored. Doyle and Roman (1982) reported a 58% carrier state in feces of a milking herd in the USA. Oosterom et al. (1982) in a survey in Holland demonstrated that 5.5% of 200 cows carried C. jejuni in the intestinal tract. 4:5. Water

Outbreaks of Campylobacter enteritis have been associated with contaminated water. Campylobacter jejuni has been isolated from estuarine, river and surface water

(Knill et al. 1978) suggesting the possibility of

transmission of the pathogen to humans and animals. A

presumptive water-borne outbreak of C .jejuni enteritis in Sweden involving about 2000 cases was reported by Mentzing (1981). Circumstantial evidence pointed to a common

untreated water supply as the source of infection.

Campylobacter jejuni was recovered from 85% of patients although contamination of drinking water was not confirmed by isolation of the organism. Effluent from a flock of

20,000 laying hens, upstream from the area from where the

subject population obtained its water supply was assumed

to be the source of contamination.

Vogt et al. (1982) repor ted a diarrheal illness in

Vermont, involving 3000 pati ents during a 2-week period,

Unfiltered and untreated wate r supplied by the common town

system was responsible for infection. Palmer et al.

(1983) identified the same biotype of C. jejuni from infected patients and their water supply, confirming the relationship between contaminated water and an outbreak of campylobacteriosis.

Sacks et al. (1986) documented a 56% gastroenteritis attack rate in residents consuming city water in

Greenville, Florida. This value compared to an attack rate of only 9% among residents of homes not supplied by city water. The common water source was from a deep well system with an open top treatment tower. Birds were costantly seen perched on the tower presumably contaminating the water. Campylobacter j e juni was isolated from 11 of 72 patients with diarrhea and in addition from 37% of 38 birds trapped in the vicinity of the well. It is necessary however to identify the respective serotypes of C. jejuni isolates derived from human cases and birds before concluding that free living avian species are responsible for contamination of water supplies.

Taylor et al. (1983) reported outbreaks of C. jejuni enteritis associated with consumption from mountain streams in the Grand Teton National Park, Wyoming. The report indicated that C. jejuni was isolated from 24% (21 of 87) and 23% (41 of 178) of patients with diarrhea during the summers of 1980 and 1981 respectively. Approximately 60% of patients with confirmed C. jejuni infection had consumed water from mountain streams during a 2 week period prior to the onset of illness.

Campylobacter jejuni was isolated from 2% of the 586 water samples. Chapter 5

OTHER MODES OF TRANSMISSION

5:1. Direct transmission

Epidemiological studies suggest the possibility of direct transmission of C. jejuni. Pai et al. (1979) documented the isolation of C. jejuni from 43% of 1004 diarrheic children in a Canadian hospital. Spread of C. jejuni infection among 72 household contacts was documented in 6 of 24 families studied. Five children and 4 adult contacts of 18 patients excreted C. jejuni in feces while 5 children and 3 adults showed gastro­ intestinal involvement. A common source of infection was responsible for symptoms in 3 of 4 members of one family who excreted C. jejuni within a 3 day period. Direct transmission was suggested in a family in which 3 members out of 5 became infected after intervals of 2-4 weeks.

Blaser et al. (1981) presented evidence of direct transmission of C. jejuni in two families. The first case involved a 2 year old male child who developed bloody

39 diarrhea and fever. Fecal cultures yielded C. jejuni and

the patient recovered following erythromycin therapy.

Within a period of 20 days 3 members of the family developed similar symptoms. Fecal cultures of 2 of the

contacts yielded C. jejuni and all 3 recovered following

administration of antibiotics. In a second case, a 30

year old man with a 9 day history of copious diarrhea accompanied by fever, infected two family contacts who developed similar symptoms within 3 days and a third 9

days after the initial onset of illnesses. Facal samples

from all patients yielded C. jejuni. It was concluded

from the course of illness that direct transmission of C. jejuni occured.

Neither vaginal nor coital transmission of C. jejuni have been demonstrated in humans (Blaser et al., 1979),

infection has been reported among homosexual men. Quinn

et al. (1980) documented 5 cases of proctitis from which

jejuni was isolated. In a typical case, a 22 year old male presented with a 9 day history of watery, and

sometimes bloody diarrhea and anal pain. He denied contact with pets or farm animals, had not been exposed to with persons who had a similar illness and had not consumed under-cooked or raw food. He acknowledged sexual

relationship with several men up to onset of illness. Rectal cultures which were negative for all common

pathogens yielded C. jejuni. Symptoms resolved sponta­ neously over a two weeks period.

Quinn et al. (1984) recovered C. jejuni from 10 of 158 homosexual men with gastrointestinal symptoms and 2 of 75

asymptomatic homosexual men. In contrast, none of the rectal cultures from a control group of 150 heterosexual men and women yielded C. j ejuni. In addition

campylobacter-like organisms were isolated from 26

symptomatic and 5 asymptomatic homosexual men from the same study. There was positive correlation between Si- jejuni infection and anilinction in the affected individuals.

In contrast to the findings of Quinn et al. (1984),

McMillan et al. (1984) documented the results of a serological study to indicate that C. jejuni infection in homosexual men is not significantly higher than in a cohort group of heterosexual men. Serum antibody titers of >40 were detected in 10.2% of 187 homosexuals and 8.9 % of 169 heterosexuals. 5.2. Perinatal transmission

Maternal systemic bacteremia can lead to fetal C. jejuni infection (Blaser et al., 1983b). Thomas et al. (1980) document a case involving a 12 day old male infant who developed C. jejuni meningitis. It was disclosed that the mother had experienced transitory diarrhea during the

7th month of pregnancy. High maternal IgG antibody was demonstrated against the strain of C. jejuni isolated from

the infant. Gribble et al. (1981) reported a case of fetal death attributed to C. jejuni bacteremia. The mother who presented during the 18th week of gestation,

reported chronic diarrhea of 3 weeks duration.

Campylobacter jejuni was isolated from maternal blood, placenta and fetal spleen.

Vesikari et al. (1981) reported a case in which a mother became ill two days after delivery. The baby developed diarrhea the following day and stools yielded C. jejuni on culture. Both mother and child developed antibodies to the same strain of C. jejuni, suggesting that transmission occured following fecal contamination of the birth canal. In a neonatal nursery in Canada, 4 patients developed

C. jejuni enteritis within 5 days. Investigations suggested that neither cross-contamination nor a common source of infection was likely (Karmali et al., 1984).

Since the serotypes of C. jejuni isolated from feces of 3 of the mothers were identical to those isolated from their infants, it was concluded that neonatal transmission of infection had occured.

Youngs et al. (1985), documented 3 cases of neonatal enteritis caused by C. jejuni. All 3 infants showed mucoid or bloody diarrhea within 72 hours after birth. The mothers had presented with diarrhea either at delivery or during the following day. In all 3 cases, C. jejuni was isolated from feces of both mother and infant. The authors suggested that Campylobacter enteritis should be considered in neonates presenting with bloody diarrhea.

DiNicola (1986) reported isolation of C. jejuni from feces of a 3-day old male infant whose mother had intermittant bloody diarrhea 5 days prior to and 4 days after delivery. Three other family contacts had intermittant diarrhea during the period 2 weeks prior to and 1 week after the infant's birth. Diarrhea ceased after administration of erythromycin. 44

5:3. Transmission by vectors

Umunnabuike and Irokanulo (1986) documented isolation of C. jejuni from adult American and oriental cockroaches.

The gut contents of 3 specimens and the surface of 1 of 690 adult cockroaches yielded C. jejuni. Notwithstanding

the low isolation rate (0.5%), the results suggest the possible role of insects in C. jejuni contamination of food products.

Rosef and Kapperud (1983) isolated Campylobacter spp.

from houseflies (Musca domestica) captured in the vicinity of poultry farms in Norway. Shane et al. (1985)

infected houseflies with C. jejuni and placed them in isolation units with specific pathogen free chicks. The organism was recovered from the feet of 20% and from the viscera of 70% of flies. Cloacal samples of subject chickens yielded C. jejuni 7 days post infection demon­ strating the potential for dissemination of campylo- bacteriosis by flies. Chapter 6

PATHOGENESIS IN MAN

6:1. Clinical course

Butzler and Skirrow (1979) reviewed the clinical manifestations of intestinal campylobacteriosis in humans.

Infections with C. jejuni do not always produce overt

clinical signs, asymptomatic carriers and a transitory state of infection are frequently encountered. Geriatric,

pediatric and immunosuppresed patients are most

susceptible and deaths are usually associated with these risk groups.

Practically all patients with campylobacter infection show some form of diarrhea. In 90% of patients the attack

is of sudden onset and usually less than a week in duration. (Blaser et al., 1979; Pai et al., 1979; Ponka et al., 1984). Feces may be bile stained, mucoid, bloody or melenic and be accompanied by flatulence. Five to ten evacuations per day during the peak of illness occurs in

50% to 60% of patients. The presence of abdominal pain is noted in the majority of patients, varying from 60% (Pai et al., 1979) to 96% (Blaser et al., 1979).

45 Fever is present in 75% to 90% of cases. Malaise, headache, nausia, emesis and anorexia are some of the symptoms associated with campylobacteriosis.

6:2. Extra-intestinal complications

Although C. jejuni is a common foodborne intestinal infection in humans, involvement of other organ systems has been reported. Thomas et al. (1980) documented a case of meningitis in a 12-day old male infant with a 20 hour history of lethargy and anorexia. It is considered significant that both the infant's mother and sister had diarrhea of three days duration about 6 weeks before delivery. Cerebrospinal fluid yielded an aflagellate strain of C. jejuni.

Urinary tract infections have been reported by several authors including Davies and Penfold (1979) who documented recovery of C. jejuni from the urine in a 77 year old patient with dysuria. The patient did not complain of diarrhea and symptoms were limited to frequent micturition including hourly nocturia. Midstream urine yielded pure cultures of C. jejuni. Serum obtained 12 days after the onset of illness showed an agglutinating titer of 1:160 to 47

the specific infective strain. A similar case described by Feder et al. (1986) involved isolation of C. jejuni

from the urine of a 6 year old girl who presented with

cloudy urine. Neither diarrhea nor fever were present and urine yielded C. jejuni on isolation. It would thus be justifiable to screen for C. jejuni in cases of chronic urinary tract infection in pediatric and geriatric patients.

It has been suggested that hemolytic uremic syndrome (HUS) may be produced by C. jejuni endotoxins. A case described by Delans et al. (1984) involved a 54 year old male admitted with acute renal failure accompanied by abdominal pain, diarrhea and emesis. Campylobacter jejuni was isolated from feces but urine and blood cultures were negative. Fumarola et al. (1985) suggested that lipopolysachcharide endotoxin of C. jejuni was one of the causes of HUS. Fatal HUS in a 4 month old male with concurrent enteritis was reported by Hag et al. (1985). In this case C. jejuni was isolated from the feces and the antibody titer against the autogenous C. jejuni strain was elevated. Other frequently encountered pathogens such as

Salmonella spp., Shigella spp., Vibrio spp., enterotoxic or enteropathogenic E. coli were absent. Guillain-Barre' Syndrome (GBS) has been associated with C . jejuni infection by several workers including

DeBont et al. (1986). These authors documented a case involving a 30 month female infant who developed GBS 9 days after onset of bloody diarrhea which yield C . jejuni.

Although diarrhea resolved spontaneously, limb paresis and facial paralysis supervened. Administration of erythromycin was initiated during the second month of illness in response to intermittant diarrhea associated with C . jejuni cultures. Recovery followed initiation of antibiotic therapy.

Septicemia is a frequent complication of C. jejuni infection. Pepersack et al. (1979) described a case involving possible transmission of C. jejuni following blood transfusion in a 67 year old patient. Septicemia was confirmed by C. jejuni positive blood cultures. When subsequently tested, the blood donor demonstrated antibodies against the same serotype of C. jejuni suggesting hematogenous transmission.

Fetal loss in a 23 year old woman was reported by

Gilbert et al. (1981) in which C. jejuni septicemia was regarded as the etiology. The patient was admitted during the 14th week of gestation showing malaise and fever with blood cultures positive for C. jejuni. Dhawan et al. (1986) documented septicemia in a study which included 33 patients with C. jejuni enteritis. As

36% of patients were under 12 months of age and 24% older than 50 years, it was concluded that extra-intestinal infection is a specific problem in pediatric and geriatric patients.

Persistent C. jejuni infection in an immunocompromised patient was described by Johnson et al. (1984). A 26 year old woman demonstrated recurrent bacteremia and enteritis caused by a single serotype of C. jejuni over a period of twelve months. The isolate was sensitive to the bactericidal activity of serum derived from a clinically unaffected subject, but not to the patient's serum suggesting an immune deficiency. Neither IgA nor IgM were detected in the patient's serum and IgG titer was elevated only after 7 months compared to a normal response within

15 days. In this case relapse rather than reinfection occured due to suboptimal and delayed antibody response.

Blaser et al. (1986), suggested that serum sensitivity indicates whether a strain of C. jejuni will cause diarrheal illness or extraintestinal symptoms. Intestinal strains are usually sensitive to serum in contrast to extraintestinal strains. Immunocompromised patients are at risk of generalized infection with C. jejuni, which can 50 be regarded as a potential systemic pathogen resulting in conditions other than enteritis (Table 5). Table 5: Extraintestinal C.jejuni infections

Guillain-Barre' Constant et al. 1983 England

Syndrome Pryor et al. 1984 Australia

Davidson & Abbott 1987 Australia

Bacteremia Franciola et al. 1985 Swi tzerland Arthritis Kowalec et al. 1980 US

Bekassy et al. 1980 Sweden

Ebright & Ryan 1984 US

Peritonitis Kristinsson et al. 1986 England

Polyneuritis Wroe & Blumhardt 1985 England

Myocarditis Florkowski et al. 1984 New.Zealand

Meningitis Goosens et al. 1986 France

Iida et al. 1986 Japan

Abortion Simor et al. 1986 Canada

Circadian Urticaria Bretag et al. 1984 Australia

Chestwall abscess Muytjens & 1982 Netherlands

Hoogenhout Chapter 7

ANTIGENIC COMPONENTS

Smith (1977) demonstrated the importance of bacterial surface structure in overcoming host defences and pathogenicity. The cell surface is the major antigenic component of bacteria, since it comes into direct contact with host cells. Campylobacter jejuni shows diverse antigenic characteristics with more than 60 serotypes classified on the basis of heat stable 1ipopolysachcharide antigens (Penner and Hennessy, 1980) and 50 serotypes differentiated by heat-labile flagellar antigens (Lior et al., 1982).

7:1. Surface proteins

Logan and Trust (1983) identified a surface protein obtained by glycine buffer extraction. This protein of molecular weight 31 Kd, was common to many strains of

£• 7e3uni• A second outer-membrane protein of 45 Kd has been identified in numerous strains.

52 7:2. Heat-stable antigen (Lipopolysachcharides)

The lipopolysachcharides (LPS) of C. jejuni consist mainly of low molecular weight lipoligosachcharides (Naess and Hofstad, 1984). These are similar to the LPS described in Neisseria spp. but differ in the fact that the molecular weight LPS of C. jejuni are antigenically diverse (Walker et al., 1986).

Eldrige, Fox and Jones (1983) subjected the outer membrane proteins of C. jejuni to sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Hemagglutinating activity was present in bands in the 16 to 25 Kd range. Lipopolysachcharides extracted using 45% aqueous phenol solution yielded a single band in the 10 to 25 Kd range.

Type-specific hemagglutination activity was detected in the elute. The authors concluded that the 10 to 25 Kd LPS heat-stable antigen, form the basis of the Penner serotyping system. This was supported by the work of Burans, Stewart and Bouigeois (1983) who demonstrated the heat-stable antigen to be of molecular weight 14 to 21 Kd and consisting of short chains without significant O-side chain branching. 7:3. Heat-labile antigen (Flagellar Protein)

The flagellum of C. jejuni, which serves as an organ of motility, is an important antigenic component (Logan and

Trust, 1983). Flagellar protein have a molecular weight ranging from 57 to 66 Kd and are collectively referred to as the 62 Kd protein. The flagellum is important in mucus colonization and hence pathogenicity and antigenicity.

(Newell et al., 1985). This is supported by the presence of antibody to C. jejuni flagellin in convalescent human serum (Newell et al., 1984).

Wenman et al. (1985) compared the antigenic profiles of three strains of C . jejuni with aflagellate mutants obtained by ultra violet mutagenesis. Although the three strains of origin were serotyped as Lior 5,6 and 7 respectively, none of the mutants were typable applying the Lior system. Thus the flagellar protein was identified as an essential determinant of the typing system based on heat-labile antigen. The authors confirmed the 62 Kd protein to be the flagellar antigen specific to Campylobacters. Sera of convalescent patients yielded several campylobacter proteins in contrast to sera from unaffected controls. The most consistent immunogen demonstrated in infected patients was the 62 Kd protein, which has potential as a vaccine against Campylobacter spp.

The antibody produced against flagellae of one serotype cross reacts with flagellar antigens of other serotypes.

This necessitates adsorption of heterologous proteins when applying the Lior serotyping technique (Lior et al.,1982). Chapter 8

MECHANISM OF PATHOGENESIS

After ingestion, an organism enters the potentially hostile environment of the intestinal lumen. In order to survive, C. jejuni must overcome host defence mechanisms

and locate a suitable site for adhesion and multiplication.

Many factors influence the virulence of C. jejuni and

other enteropathogens. Attachment to the mucosal surface, colonization of the mucosa and invasion, are three sequential stages required for infection.

8:1. Mucosal adherence and colonization

A series of experiments conducted by Newell et al. (1985a) demonstrated the role of the flagellum after

jejuni enters the intestinal lumen. Colonization of intestine of 4 to 5 day old mice by a clinical isolate of

C. jejuni and its aflagellate variant were compared. The

56 isolate colonized the gastrointestinal tract successfully, as demonstrated by an increase in number of organisms recovered compared to the infective inoculum. Higher numbers of organisms were obtained from the lower small intestine, cecum and colon compared to the stomach and duodenum. Feces yielded C. jejuni for up to 31 days post-infection.

In contrast, the aflagellate C. jejuni variant did not colonize the intestinal tract to the same extent. The number of organisms recovered was not significantly greater than the inoculum. Feces was negative for

C. jejuni after 7 days of infection. The authors concluded that the flagellum plays an important role in attachment and colonization and contributes to the virulence of C. jejuni.

Isolation of C . jejuni from both healthy and experimental animals is an indication of successful colonization and attachment of the organism in the gastrointestinal tract of the host (Butzler and Skirrow,

1979; Manninen et al., 1982). Dijs and de Graaf (1982) demonstrated the absence of adhesive structures such as pilli on the surface of C. jejuni by staining organisms with phosphotungstic acid and platinum prior to examination using an electron microscope. Elaboration of an adhesin by C. jejuni has been

demonstrated (Lastovica, 1983; Naess et al., 1983). The property of adherence has been shown by Taylor, Ackerman and Fox (1985), to be correlated with pathogenicity.

Fifteen isolates from clinical cases of enteritis and 21

isolates from asymtomatic carriers were radiolabelled with [3H]alanine and [3H]leucine and cultured in chick embryo

and HeLa cells. Isolates from patients with enterocolitis had a greater tendency to adhere to host cells compared to C. jejuni organisms derived from unaffected carriers.

Virulence is dependant on successful colonization of mucus and attachment to intestinal mucosa, a property which is facilitated by functional flagellae (Gianella, 1981).

8:2. Mucosal invasion

Levine et al. (1983) catogorized enteric pathogens into five groups depending on invasive properties.

1. Mucosal adherence and production of an enterotoxin

2. Mucosal adherence and dissolution of the enterocytic

brush border 3. Mucosal invasion and intra-epithelial cell proli feration

4. Mucosal translocation followed by bacterial

proliferation in the lamina propia and mesenteric lymph nodes

5. Mucosal translocation followed by systemic infection.

Campylobacter jejuni is responsible for clinical changes attributed to three of these mechanisms.

a. Mucosal adherence and enterotoxin production causes acute watery diarrhea.

b. Mucosal invasion and epithelial cell proliferation results in bloody diarrhea with the presence of inflammatory cells in feces. Cellular damage and necrosis occurs in the terminal ileum and the colon (Walker et al., 1986). Blaser et al. (1980b) demonstrated inflamatory infiltrates in the lamina propia and crypt abscesses, similar to the lesions produced by Salmonella spp.and

Shigella spp. These changes may be detected in the tissues obtained by rectal biopsy as documented by Price et al.,(1979) and Duffy et al.,(1980). 60

c. Campylobacter jejuni has the ability to translocate to the mesenteric lymph nodes in gnotobiotic mice (Youssef et al.,1985).

Mucosal translocation and subsequent proliferation of bacteria in the lamina propi a results in clinical changes ranging from severe dysente ry to hematochezia. Skirrow (1977) described a case of mesenteric lymph node involvement in a 33 year old female patient with profuse diarrhea. An emergency lapa rotomy was performed due to acute abdominal pain ascribed to perforation of the bowel,

On examination, the ileum was found to be severely inflamed and edematous. Seve ral large 'fleshy' mesenteric lymph nodes were present and C. jejuni was isolated from feces.

Phagocytosis may influence the virulence of C. jejuni.

Kiehlbauch et al. (1985) showed that, the organism may survive within the cell for up to 7 days and subsequently exert a pathogenic effect similar to Salmonella typhi.

Invasiveness of C. jejuni in- vitro was demonstrated by Moyen et al., 1985) using a HeLa culture system. Cells were incubated with 10 organisms for 1 hour at 37°C in an atmosphere containing 5% carbon dioxide. Invasive strains of C. jejuni were located within 10% or more cells with at 61 least 20 bacteria per cell. Of the 40 strains derived from humans, 23 (58%) were non-invasive, 11 (28%) were invasive and 6 (14%) were intermediate.

Isolates of C. jejuni may exhibit one or more characteristics such as adherence, invasiveness, intracellular proliferation and translocation. Virulence and clinical response will depend on the specific characteristics of the isolate and the extent to which these properties are expressed. Chapter 9

SEROTYPING

In 1957 King documented the isolation of pathogens referred to as 'related vibriosThese organisms obtained from four cases of human enterocolitis demonstrated some morphological characteristics in common with Vibrio fetus.

Since this time progress has been made in the taxonomy and definition of biochemical criteria of the group, later to be assigned to the new genus Campylobacter. The first attempt to classify Vibrio fetus, using serological techniques, was reported by Berg et al. (1971). These authors extracted heat labile and heat stable antigens from Vibrio strains derived from 6 outbreaks of enterocolitis. Antigens were inoculated into rabbits to produce reference antisera which were then used to develop a serotyping scheme. Though both heat-labile (H) and heat-stable (0) antigens were evaluated, only heat stable antigen was used in a combined sero-biotyping scheme, since the 'H' antigen reacted to more than one antiserum.

Three distinct serotypes and two biotypes were recognized, forming the basis of an identification system. The five catogories of Vibrios which were identified by the sero-biotyping system comprised:-

62 Group 1: Serotype A-Biotype I (A-I),

Glycine negative, hydrogen sulfide negative.

Group 2: Serotype A-Bio subtype I (A-sub I),

Glycine negative, hydrogen sulfide positive

Group 3: Serotype A-Biotype II (A-II),

Glycine positive, hydrogen sulfide positive

Group 4: Serotype B (B).

Group 5: Serotype C (C).

Berg et al. (1971) related these serogroups to bio­ chemical characteristics and the clinical manifestations in various species. Group A-I and A-sub I were responsible for vibrionic infertility in cattle and were not able to survive in the intestinal tract. Group A-II,

B and C were associated with abortion in sheep but could tolerate an intestinal environment.

Comparison between the type species Vibrio cholera isolated by Pacini in 1854 and the 'related vibrios' described by King in 1957 confirmed the biochemical differences between the groups, notwithstanding morphological similarities.Campylobacter was advanced as a new genus by Sebald and Veron (1963). The principal differences between Campylobacter spp. and Vibrio spp. as defined by Veron (1982) are summarized in Table 6.

Following the investigation of Veron and Chatlain (1973), it was accepted that group C vibrios described by Berg et al. (1971) were C. jejuni.

Butzler and Skirrow (1979) serctyped Campylobacter isolates using both heat labile and heat stable antigens. Six serotypes were identified among 90 isolates. In addition Campylobacters could be differentiated into two biotypes on the basis of hydrogen sulfide production.

Abbott (1980) used the 'O' and 'H' antigens to serotype six strains of C. jejuni using a tube agglutination method. Lauwers (1981) employed a plate agglutination technique to identify 16 serogroups among 300 isolates.

A major advance was represented by the studies conducted by Penner and Hennessy (1981) and Lior et al in (1982) who independently developed serotyping schemes.

These identified specific antigens and facilitated the recognition of a greater number of serotypes of C . jejuni.

Penner and Hennessey (1981) employed a passive hemagglutination procedure in which a saline extract was Table 6. Differences between the genera Vibrio and

Campylobacter

Characteristic Vibrio spp. Campylobacter spp. (Pacini, 1854) (Sebald and Veron, 1963)

Morphology curved or straight curved or helicoidal

Coccoid forms occasional frequent

Respiration aerobic microaerophilic Glucose fermentative not metabolized

metabolism (without gas)

Source: Veron, 1973. prepared from the soluble, heat stable 'O' antigen comprising the surface lipopolysachcharides (LPS). Antisera to these antigens produced in New Zealand white

rabbits were used to identify 23 serotypes from among 91

isolates of C . jejuni. About 60 serotypes have now been

identified by the Penner method (Penner et al., 1983).

Lior et al. (1982) identified 21 serotypes from a

collection of 815 isolates using heat labile 'H' antigens.

These flagellar antigens derived from formalinized, inactivated suspensions of C. jejuni were used by Lior et al. (1982) to prepare a range of antisera in New Zealand white rabbits. The polyclonal antisera were adsorbed with homologous heat stable and heterologous, cross reactive antigen suspensions. The Lior serotyping system, which is read using slide agglutination, can be used to identify over 50 serotypes.

A comparison between the Penner and Lior systems conducted by Patton et al. (1985) indicated that the

Penner system is marginally superior in specificity. A total of 96.1% of 1405 isolates could be typed using the Penner system in comparison to 92.1% with the Lior method.

A disadvantage of the Penner system is that a small fraction of antigens may react with multiple antisera.

The method does not require adsorption of heterologous antisera. 67

Of the human and non-human isolates, which have been routinely typed using the Penner system, approximately 50% react to only one antiserum. Approximately 30% react with two antisera while the remainder (20%) react with three or more antisera. In the majority of multiple reactions, one antiserum predominates with weak reactions associated with the second or third antisera. Generally when conducting epidemiologic investigations, isolates reacting with more than one antiserum are assigned to a type corresponding to the strongest reacting antigen. Multiple reactors are designated as 16(1), with 16 as the primary antigen and 1 as the weak secondary reaction. In most instances multiple reactions occur in specific combinations such as 36(23), 8(17), 44(1).

The method of Lior identifies a larger proportion of

C. jejuni antigens which react to single antisera, but the technique has the disadvantage of requiring a multiple absorption process. Though the Lior method is simpler and involves less expensive equipment, the Penner system is considerably faster for diagnostic laboratory appli­ cations . Chapter 10.

TOXIGENICITY

10:1. Introduction

Campylobacter jejuni has been isolated from both healthy individuals (Rajan and Mathan, 1982) and from diarrheic patients (Stoll et al., 1982; Bokkenheuser and Sutter, 1981). Companion animals, especially dogs and cats may serve as healthy carriers of C. jejuni (Hosie et al., 1979), but a higher prevalence is noted in diarrheic animals (Ferreira et al., 1979). Campylobacter jejuni has been isolated from clinically normal chickens (Simmon and

Gibbs, 1979), cattle (Bryner et al., 1972), zoo animals

(Luechtefeld et al., 1981) and laboratory species (Fox, 1982) .

The isolation of C. jejuni from both healthy and clinically affected humans and animals presupposes the existence of virulent and non-virulent strains amongst the isolates. Determination of the virulence of C. jejuni isolates obtained from humans and animals has included

68 animal models, iji vivo trials and jji vitro studies involving cell-association and penetration (Newell et al.,

1985) .

Reports relating to quantitative assay of specific toxins produced by isolates of C. jejuni are limited.

This contrasts to the research on quantifying the enterotoxins of V. cholera and E. coli (Yolken et al., 1977; Back et al., 1979; Sack et al., 1980; Bongaerts, 1985) .

10:2. Toxins

Both an enterotoxin and a cytotoxin have been identified from C. jejuni. The enterotoxin is considered an endotoxin while the cytotoxin is an exotoxin. Campylobacter jejuni strains may produce either, both or neither of these toxins. Production of toxins in-vitro depends on provision of suitable media and growth conditions. 10:2(1). Enterotoxin

Ruis-Palacios et al. (1983) demonstrated a cholera­ like enterotoxin from 24 out of 32 isolates of C. jejuni derived from diarrheic patients using the Chinese Hamster

Ovary (CHO) cells and the Cyclic Adenosine Mono-Phosphate (cAMP) assay. Of 6 strains isolated from carriers, only 1 produced the toxin. The author further characterized the toxin as heat labile at 56°C for 1 hour with a molecular weight of 10 to 100 Kd and was inactivated by pH below 2 and above 8. McCardell et al. (1984a) reported a similar finding using ELISA, CHO and adrenal tissue (Yl) cell assays. 10:2(2). Cytotoxin

In 1983 Yeen et al. demonstrated a toxin produced by C . jejuni which caused destruction of HeLa cell in 24 to 48 hours. The toxin caused cell rounding, nuclear pyknosis, loss of cell adherence in monolayers and death and floatation of cells. Since sonicated bacterial cells did not show toxin activity, the authors suggested that death of host cells was caused by an exotoxin and not an endotoxin. The toxin was shown to be heat labile at 100°C for 30 minutes. Johnson and Lior (1984) documented similar findings using Vero cells. They were able to show cytotoxic activity in more than 60% of the 80 isolates derived from fecal samples in contrast to cytotonic

(enterotoxic) activity in more than 85% of the isolates.

Goosens (1985) used CHO cells to demonstrate cytotoxins produced by 33 out of 39 C. jejuni strains isolated from patients with watery stools.

McCardell et al. (1986) characterized the cytotoxin to comprise of two units. Cytotoxin 1 has a molecular weight of 70 kd and an isoelectric point of 9.0. This unit was heat stable at 100°C for 5 minutes, was trypsin resistant (1 mg/ml/hr at 37°C) and caused hemagglutina­ tion but not hemolysis in rabbit erythrocytes. The second unit, cytotoxin 2, was trypsin sensitive, heat labile and hemolyses rabbit erythrocytes.

Enterotoxin produced by C. jejuni has similar character

istics to the toxins of Vibrio cholerae and E. coli. All three toxins are heat-labile, elevate cAMP production, elongate CHO cells and neutralize anti-cholera toxin serum

(McCardel et al., 1984b). Due to the similarity between the enterotoxins of C. jejuni and E. coli both in respect to antigenicity and physical properties, most assays to quantify the enterotoxins of E. coli or V. cholerae may be

applied to C. jejuni.

10:3. Assays

10:3(1). Enzyme Linked Immunosorbent Assay

Holmgren et al. (1973) reported that a specific glyco-

lipid, ganglioside GMl, contains a receptor structure which fixed cholera toxin in a double-diffusion gel

precipitation technique. Due to the specific fixation of

enterotoxin, ganglioside GMl was subsequently used to bind

cholera toxin in a modified enzyme linked immunosorbent

assay (GM1-ELISA) as shown in Figure 4. 73

Svennerholm and Wiklund (1983) describe a GMl-ELISA in which microtiter wells were coated with GMl to bind E. coli enterotoxin, thus increasing the sensitivity of the assay. Bongaerts (1985) effected modifications to the

GMl-ELISA to expedite the assay to allow completion within 8 hours.

Enterotoxin of C. jejuni was shown to be immuno- logically similar to cholera toxin by McCardell et al.

(1984b) and to E. coli enterotoxin by Klipstein and Engert

(1985). Thus GMl-ELISA can be used to assay C. jejuni enterotoxin.

Ruiz-Palacios (1985) and Klipstein(1986) have success­ fully applied the GMl ELISA modification to assay heat-labile enterotoxin of C. jejuni.

The following reactions occur during GMl ELISA:

a. GMl fixes LT (labile toxin of C. jejuni)

b. LT binds CT-IgG (goat serum against cholera toxin)

c. CT-IgG is bound by horseradish peroxidase conju­ gated anti-goat serum (in rabbits)

d. Horseradish peroxidase reacts with O-phenyldiamine

dihydrochloride substrate to produce a color

reaction. If a culture filtrate from a C. jejuni isolate contains a labile toxin, the positive color reaction can be

detected and quantified using a spectrophotometer.

10:3(2). Cell culture assay

Cell culture assays have been used extensively to

detect enterotoxins of V. cholera and E. coli. Donta et al. ( 1974 ) used adrenal cell lines (Yl cells) for detection of E. coli enterotoxin while Guerrant et al

(1974) employed Chinese hamster ovary cells (CHO) for

studies on V. cholera and E. coli toxins. Speirs et al.

(1977) assayed the enterotoxin activity of E. coli using Vero cells. Gubina et al. (1980) and Ruiz Palacios, (1983) quantified the enterotoxins of C. jejuni using CHO

cell lines.

Two approaches have been used to assay enterotoxins

using CHO cells.

a. Cyclic Adenosine Monophosphate assay:

Schaffer et al. (1970) reported an elevation in the

concentration of adenosine 3':5'~ cyclic monophosphate (cAMP) in the intestinal mucosal cells of rats after

treatment with cholera toxin. Similar results were

reported by Bourne et al. (1973) who demonstrated an

accumulation of cAMP in human leukocytes caused by cholera enterotoxin, in contrast to other agents such as

prostaglandin-El. Guerrant et al. (1974) evaluated the

effect of enterotoxins in-vitro by incubating CHO cells with either cholera toxin or E. coli heat-labile toxin.

The cAMP concentrations were assayed by the procedure of Gilman (1970), who reported a significant increase in cAMP levels by both toxins. An elevation in level of cAMP over

controls indicated the presence of an enterotoxin.

b. Cell Morphology Assay:

Hsie et al. (1971) and Hsie and Puck (1971) noted morphological changes in CHO cells, produced by cAMP. Affected cells showed bipolar elongation and the number of cells showing changes was proportional to the concentration of cAMP. The cAMP effect on cells, which is opposite to the response stimulated by malignant viruses or carcinogenic agents, is called reverse transformation.

Puck et al. (1972) detailed the mechanisms by which cAMP affected the integrity of membranes in mammalian 76 cells. Knob-like structures on the cell membrane are lost within 15 minutes and the membrane becomes smooth. Cyclic AMP causes organization of the microtubular microfibrillar (MT-MF) system resulting in an increase in microtubules which become arranged into parallal arrays.

Following this stage the cell elongates to assume a fibroblast like morphology. Elongation is visible after 5-8 hours depending on the concentration of cAMP.

Ruiz-Palacois et al. (1983) reported both elongation and increased cAMP production in CHO cells when incubated with 24 - 36 hour cultures of C. jejuni obtained from patients with diarrhea. A total of 75% of the 32 isolates from diarrheic patients showed enterotoxin activity denoted by morphological and cAMP assay. Only 1 of 6 isolates from non-affected carriers showed any detectable toxin activity. Elongation of CHO cells, which indicates the presence of enterotoxin may be used to characterize isolates of C. jejuni. Chapter 11.

CONCLUSION

Campylobacter jejuni is one of the significant pathogens causing enterocolitis in humans. Due to microaerophilic and thermophilic requirements and the prolonged lag phase of growth, C. jejuni was relatively obscure prior to the late 1970's. The development of selective media and microaerophilic gas systems, facilitate isolation and propogation of C. jejuni which is now frequently recovered from man and animals. In order to understand the epidemiology of campylobacteriosis and the zoonotic implications of C. jejuni, extensive investi­ gations have been initiated during the past five years.

Campylobacter jejuni may be isolated from affected human and animal patients, in addition to healthy individuals who maintain the organism in the intestinal tract. The prevalence of C. jejuni in humans is higher in third world nations than in developed countries and the organism is more frequently isolated from patients with gastrointestinal conditions (Table 7).

77 The prevalence of C .jejuni in the human population may range from 0 to 31% depending on the age of the population

surveyed, country, socio-economic conditions and inter­

current disease or parasitism. Isolation of C . jejuni

from non-diarrheic patients is not unusual and frequently the organism cannot be recovered from feces of a patient with gastrointestinal signs. Detecting an increase in circulating antibody titer is a reliable indication of

infection.

Infection with C . jejuni may be asymptomatic or produce a wide range of changes including watery diarrhea, severe enteritis with ulceration of the lower intestinal tract, hematochezia and generalized malaise (Walker et al.,

1986). Clinical severity depends on the elaboration of toxin by the strain infecting the patient. Some serotypes of C. jejuni produce an enterotoxin which is chemically similar to the toxin of Vibrio cholerae causing profuse diarrhea while others elaborate a cytotoxin which induces a local inflamatory reaction resulting in bloody or mucoid diarrhea. Many strains are known to produce both toxins. The severity and nature of campylobacteriosis depend on the degree of expression of these two toxins.

Campylobacter jejuni is also associated with several systemic infections such as meningitis, myocarditis, arthritis, nephritis and conditions such as hemolytic 79

Table 7: Prevalence of C. jejuni in specific populations

Asymptomatic Diarrheic

individuals patients

Third World 1 1 nations | 5-17% | 8-31%

1 1

Developed 1 1 nationss | 0-1% | 4-7%

1 1 uremic and Guillain-Barre' syndromes (Walker et al., 1986).

Avian species, including commercial poultry have been identified as an important reservoir of C. jejuni as

denoted by isolation rates of up to 90% in layer and

broiler flocks and 100% in turkeys. Intensive commercial management of poultry, has contributed to the maintenance of a reservoir state which has significant human health

implications. Although C. jejuni is reported to cause hepatic lesions in mature chickens, infection is currently not regarded as a disease entity in the poultry industry.

Re-use of litter and deficiancies in biosecurity are

important in maintaining the cycle of C. jejuni infection.

Broilers which are grown on litter are more likely than commercial layers to carry C. jejuni infection at the time of slaughter.

Cross contamination occurs between infected and clean carcasses at various sites in the processing plant such as the scalding tank, defeathering machines, evisceration line and chill tank (Wempe et al., 1983). Since 70% of oven-ready carcasses and portions are contaminated, consumption of under cooked or raw chicken may lead to food-borne campylobacteriosis. Conclusive evidence of food borne campylobacteriosis is represented by a correspondence in serotype between isolates from a food source and affected patients.

Approximately 60 C. jejuni serotypes have been identified, using either the Penner or the Lior system.

Campylobacter jejuni may elaborate either or both, enterotoxins or cytotoxins. Any C. jejuni serotype may be catogorized according to the presence (+) or abscence

(-) of toxin.

Type Cytotoxin Enterotoxin

1 + +

2 +

3 + 4 -

Infection with a strain which is enterotoxin-positive and cytotoxin-negative will most likely result in watery diarrhea. A cytotoxin-positive, enterotoxin-negative strain will cause severe enteritis with hematochezia accompanied by abdominal pain. Strains with neither toxin result in asymptomatic infection in contrast to serotypes which elaborate both toxins which may evoke watery to bloody diarrhea with malaise and abdominal pain. There is no specific study on the epidemiology or prevalence of C. jejuni infection in either animal or human populations in Louisiana. The present study using both serology and toxigenecity assays was initiated to obtain an understanding of the epidemiology of

Campylobacteriosis. Chapter 12

PROCEDURES

The general objective of the research program concerned an investigation of the prevalence, serotype and pathogenicity of C. jejuni.

12:1. Prevalence study

The objective was to determine the prevalence of

C* jejuni in diarrheic patients and in avian cases submitted to a diagnostic laboratory.

12:1(1). Sampling techniques

This study of prevalence of C. jejuni in avian species comprised the cases submitted to the Louisiana Veterinary Medical Diagnostic Laboratory during the period February

1987 through July 1988. In addition, hunter-killed

Anseriformes, available in the vicinity of the Rockefeller

Refuge, Cameron Parish, Louisiana, were sampled. Cloacal

83 swabs from all cases were routinely cultured for

C . jejuni.

Fecal specimens from human patients with diarrhea comprised both admitees and outpatients at the Our Lady Of The Lake Regional Medical Center, Baton Rouge, Louisiana.

Submissions during the period August 1987 through July

1988, were routinely cultured for the presence of

C . jejuni.

12:1(2). Culture and identification of C. jejuni

Figure 3 is a diagramatic representation of the procedures to identify C. jejuni isolates.

a. Culture and isolation

The selective Campylobacter medium which was used for all isolation procedures consisted of a basal medium of brucella agar (Difco Laboratories, Detroit, Michigan), incorporating 5% ruminant blood and Butzler's Antibiotic

Supplement (Oxoid Limited, Hampshire, England). Each 3ml vial of supplement contained 12,500 units bacitracin,

25 mg cycloheximide, 5,000 units colistin sulphate, 7.5 mg cephazolin sodium and 2.5 mg novobiocin. Growth on selective medium

/ \ Yes No ----> Eliminates enteropathogens other than

I Campylobacter spp. Growth at 42° C

/ \ Yes N o ------> Eliminates C. fetus & C. pyloridis

I Nalidixic acid sensitivity

./ \

Sensitive Resistant------> Eliminates C. laridis

I Hippurate hydrolysis

/ \ Yes N o ------> Eliminates C. coli

I C. jejuni------> Toxin assay

I I / \ H S production Passive Enterotoxin Cytotoxin

/ \ Hemagglutination / \ / \

Yes No | +ve -ve +ve -ve

Biotype Serotypes 1 to 60 I II

Figure 3. Scheme to speciate and serotype Campylobacter

jejuni isolates Fecal swabs were placed in Cary-Blair transport medium

(Difco Laboratories) and processed within 24 hours. Swabs impregnated with biological material were used to inoculate plates of selective medium. Incubation was carried out at 42°C under microaerophilic conditions in a sealed bag containing a mixture of 5% oxygen, 10% carbon dioxide and 85% nitrogen (Liquid Carbonic, Baton Rouge,

Louisiana).

Following incubation for 48 hours, the plates were examined for evidence of growth. Primary cultures were recognized as flat, non-hemolytic gray colonies with spreading borders. Plates were incubated for an additional 24 hours if no visible growth was present.

b. Gram staining

A sterile platinum wire loop was used to transfer a portion of a colony to a drop of sterile water on a glass microscope slide. After mixing, the suspension was allowed to dry in air and the preparation was stained with a modified Gram stain procedure, substituting basic fuchsin for safranin as a counterstain. Slides were also stained directly with basic fuchsin for rapid determination of morphology. 87

c. Motility

Cultures of presumptive Campylobacter spp. were incubated in brucella broth for 20 hours at 42°C. A 0.1 ml

aliquot of the culture was tranferred to a microscope slide using a sterile pipitte and examined under dark

field illumination. Characteristic rapid darting movements indicated the presence of Campylobacter spp.

12:1(3). Biochemical characteristics used in identi­

fication and biotyping (Table 8).

a. Nalidixic acid sensitivity

A colony from the primary culture was streaked onto a blood agar plate with a 30 microgram nalidixic acid Sensidisc (BBL Microbiological System, Cockeysville, MD).

After incubation at 42°C for 24 hours, cultures were examined to determine sensitivity.

b. Hydrogen sulfide production

Hydrogen sulfide production was determined according to the procedure described by Lior (1984). A semisolid agar base medium containing brucella broth, sodium hypo- phosphate, potassium hypophosphate, ferrous sufate, sodium metabisulfite and sodium pyruvate was prepared, dispensed into 3-ml aliquots in sterile 13 X 100 mm screw cap tubes

and stored under refrigeration. Campylobacter jejuni test

cultures were streaked for contiguous growth on blood agar plates. Using 24-hour cultures, a 10 ul disposable

plastic inoculating loop was repeatedly drawn across the

surface of the plates to gather a 5 mm spherical mass of

bacteria. This inoculum was suspended, without mixing, in the upper third of the medium. Inoculated tubes were incubated in a 37 °C water bath for 2 hours, then examined.

A blackening of the bacterial mass indicated sulfide production. Campylobacter jejuni Lior serotype 6 served as a positive control.

c. Hippurate hydrolysis

Colonies from a secondary culture were mixed with 2 ml sterile water in a tube. A hippurate disc (1.6 g sodium hippurate) as used in the Minitek Identification System (BBL Microbiological System, St.Louis, Missouri) was added to the suspension and the mixture was incubated at 37°C for 2 hours. An aliquot of 1 ml ninhydrin solution prepared by rehydrating 3.5 g crystalline ninhydrin (Sigma

Chemical Co.) in 100 ml of an equal mixture of acetone and butanol was added to the incubated solution. After 2 hours at room temperature, a positive reaction was denoted by the development of a blue color in the solution. Table 8. Differences in characteristics of catalase

positive Campylobacter spp.

Species Optimal Nalidixic Hippurate Hydrogen growth acid hydrolysis sulfide temp. sensitivity prodn.

C. fetus 25 °C R - - o

C. coli o O S - -

C. jejuni(Bio I) 42 °C S + - C. jejuni(BioII ) 42 °C S + +

C. laridis 42 °C R - -

C. pyloridis 37 °C R ? +

R: Resistant S: Sensitive

+ indicates production or reaction

- indicates no production or reaction

?: Not known

Source: Skirrow and Benjamin, 1979. 90

12:2. Serotyping

Isolates of C. jejuni were examined to determine the identity and prevalence of serotypes and to investigate whether any commonality existed between the two populations in respect of serovars.

Penner serotyping was used to characterize isolates of C. jejuni. This system is based on passive hemagglutin­ ation of heat stable, soluble antigens which can be used to produce specific antibodies in rabbits. The heat stable antigen extracted from an isolate of C. jejuni was adsorbed onto sheep erythrocytes to be agglutinated against rabbit antisera.

12:2(1). Preparation of antigen

The test isolate was inoculated on campylobacter agar plates and incubated at 42°C under microaerophillic conditions until confluent growth was obtained (48-72 hours). Growth from 4 plates was pooled to prepare antigen. A

1.5 ml aliquot of isotonic phosphate buffered saline

(IPBS) at pH 7.0 was flushed on to each plate. The

solution comprised 8g. sodium chloride, 0.2g. potasium

hydrogen phosphate, 2.9g. sodium hypophosphate, 0.2g potasium chloride and 600ul concentrated hydrochloric acid in 11 water. Colonies were transferred to a tube using a bent glass rod.

The tube containing C. jejuni in IPBS was placed in a

water bath at 100°C for one hour and thereafter was allowed to cool and then centrifuged at 10,000 rpm (12,000 xg). The supernatant was poured into a glass vial and two drops of preservative (1% thimerosal, Sigma Chemical Co.) was added to the supernatant. The heat-stable antigen was stored at 4 °C until further use.

12:2(2). Preparation of antisera

Confluent bacterial growth of C. jejuni reference strains (Penner typing) were transferred to 3 ml of 0.85% sodium chloride solution, washed twice and resuspended in saline.

After a pre-immune blood sample was obtained, New

Zealand white rabbits (3-4 Kgs) (Hazelton Research Animals, Devon, Pennsylvania) received 5 sequential intravenous inoculations of antigen at 3 day intervals.

Blood was collected by cardiac puncture 10 days after the last injection. Blood was allowed to clot and serum was separated for storage at -20°C.

12:2(3). Adsorption of antigen

Sheep blood was centrifuged and the erythrocytes were washed three times in IPBS. Erythrocytes were resuspended in IPBS at the original volume and the hematocrit value was used to determine the percentage of cells in the suspension. A 1% cell suspension was prepared by appropriate dilution with IPBS.

Antigen was diluted to 1:10 by mixing 0.4 ml of the preparation with 3.6 ml of IPBS. An equal volume of 1%

SRBC suspension was mixed with the antigen, the tubes were covered and incubated at 37°C for 1 hour and thereafter centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded, the SRBC preparation was washed and resuspended in the original volume of IPBS. This amount of antigen-coated SRBC was sufficient for one 96 well plate titration. 93

12:2(4). Serial dilution of sera

Sera were stored in undiluted form at -20°C. A series of tubes containing 1:5 dilution in IPBS was made from the

original serum for storage at 4°C with 1% thimerosal as a preservative. Serum was diluted to 1:20 with IPBS for titration.

A 50 ul dispenser was used to transfer IPBS to each

well of the microtiter plate (Dynatech Laboratories, Alexandria, Virginia). An aliquot of 50 ul of 1:20 antiserum (in IPBS) was added to the first well of each

row, yielding a 1:40 dilution of antiserum in the first well. A microtiter diluter was used to prepare two-fold

serial dilutions. The antiserum dilutions for each

antigen ranged from 1:40 to 1:1280.

12:2(5). Agglutination

Using appropriate dispensers, 50 ul of antigen- sensitized sheep erythrocyte suspension was placed in each well containing diluted antiserum. The plate was covered

to prevent evaporation of reagents and incubated at 37°C

for 1 hour. The plate was then removed and placed in a refrigerator at 4°C. A minimum of 1 hour was allowed before the plate was examined for evidence of agglutination. Plates were routinely retained overnight to permit development of color to facilitate recognition of hemagglutination.

12:3. Toxigenicity testing

The objective of this study was to investigate the toxigenicity of C. jejuni isolates of known serotype using cell culture and modified ELISA techniques. Profiles of the toxins produced by the isolates were assayed to identify the toxigenic strains prevalent in collected specimens. This data was intended to serve as a baseline for further epidemiologic investigations of campylo- bacteriosis in Louisiana. Toxigenic assays were conducted to determine whether any relationship existed between serotypes and the occurence of disease in either humans or animals. 12:3(1). Enterotoxin assay

a. Enterotoxin production

Each test strain of C. jejuni was cultured in brucella agar containing 10% ovine blood for 48 hours.

Approximately 15 CFU was inoculated into brucella broth supplemented with a 0.25% mixture each of L-asparagine,

L-serine (Eastman Kodak Co., Rochester, New York) and L-glutamic acid (Difco Laboratories). Cultures were incubated at 42°C for 24 hours in a humidified atmosphere,

followed by incubation for 10 minutes with a suspension of

500 IU polymyxin B sulphate (Pfizer Inc., New York, New

York). Cultures were centrifuged at 20,000 xg for 20

minutes at 4°C. The supernatant was filtered through a

0.22 um Aerodisc membrane (Gelman Sciences Inc., Ann Arbor, Michigan) and immediately tested for toxin production.

b. GM1 ELISA

This assay is based on the property of ganglioside GMl to bind to C. jejuni enterotoxin. Binding of toxin is detected by a modified ELISA system which requires reactions with two successive antibodies and an enzyme-substrate reaction (Figure 4). Cholera antitoxin is used as the first antibody and horseradish peroxidase conjugated anti-goat serum as the second antibody. Ganglioside Ml ELISA was performed using Bongaerts technique (1984) with modification of Clarke (1986).

A u-bottom, 96 well polyvinyl chloride microtiter plate (Dynatech Laboratories Inc.) was used for the procedure.

Wells were coated with GMl ganglioside (Sigma Chemical

Co.) by adding 150 ul (100 ug/ml in PBS) per well and incubating at 22°C for one hour.

The wells were washed three times with PBS-Tween (PBST)

(Fisher Scientific Co., Fair Lawn, New Jersey) and 200 ul of neonatal calf serum (1% in PBST) was placed in each well. The plate was incubated at 37°C for 30 minutes.

The wells were washed three times as previously described and 150 ul of test-sample enterotoxin (two-fold dilutions of culture supernatants from 1:10 to 1:100) was added to each well in each row in sequence. The plate was incubated at 22°C for 2 hours. Enzyme-Substrate reaction

Enzyme

Anti-rabbit IgG

Rabbit anti-LT

Milk protein

C. jejuni LT

Figure 3: Diagrammatic representation of the principle of GM1-ELISA. After washing, 150 ul of goat anti-CT (Jackson Immuno

Research Laboratories Inc., West Grove, Pennsylvania) was added at a dilution of 1:1000 to each well and the plate was incubated at 37°C for 1 hour in a humidified chamber.

After washing, 150 ul of horseradish peroxidase- conjugated rabbit anti-goat serum (Jackson Immuno Research

Laboratories Inc.) was added at a dilution of 1:1000 to each well. The plate was incubated at 37°C for one 1 hour in a humidified chamber.

The wells were washed and 150 ul of hydrogen peroxide -O- phenylenediamine dihydrochloride (H202-0PD)(Eastman Kodak Co.) substrate solution was added to each well and the plate incubated at 22°C for 1 hour in the dark.

To stop further reaction after the last step and to intensify color, 50 ul of sulphuric acid (H2S04, 2.5 mol/1) (J.T.Baker Chemical Co., Phillipsburg, New Jersey) was added to each well.

The optical density of the solution in each well was determined using a MR 600 microplate reader (Dynatech

Laboratories Inc.) at 492 nm. An extinction value of 0.1 indicated the presence of a significant concentration of c. CHO cell assay

Morphological changes, comprising an elongation

(bipolarity) of the rounded cells can be induced by

enterotoxin of C . jejuni, E . coli and V. cholerae. This property is used to demonstrate the presence of toxin elaborated by test isolates of C . jejuni. Supernatants of brucella broth cultures of C. jejuni were incubated with a

suspension of CHO cells and the percentage of cells which showed morphological changes was recorded.

Stock cultures of CHO cells (American Type Culture Collection, Rockville, Maryland) were incubated at 37°C, in 6% carbon dioxide in Hams F12 medium (Gibco

Laboratories, Grand Isle, New York) supplemented with 10% fetal calf serum. Cultures were passaged at interva Is by trypsinization and seeding of additional flasks.

A suspension of CHO cells was removed to estimate concentration using a hemocytometer. An appropriate dilution to contain about 5000 cells in 0.25 ml F12 medium with 1% fetal calf serum, was added to each of the 96 wells of a flat-bottomed culture plate. The supernatant from a C. jejuni culture (two-fold dilution) was added immediately to the cell suspension. Cells were examined at eight hour intervals for evidence of bipolarity and elongation. One hundred cells were counted and the percentage of elongated cells was determined. Elongation in 50% or more cells denoted the presence of enterotoxin. This assay was performed concurrently with the GMl-ELISA to detect enterotoxin.

12:3(2). Cytotoxin assay

a. Cytotoxin production

Test isolates of C. jejuni were grown on a biphasic culture system (Goossens et al., 1985). The solid phase consisted of Mueller-Hinton medium (Gibco Laboratories) with the liquid phase comprising Medium 199 (Gibco

Laboratories) with Earle's salts and L-glutamine. The plate was incubated at 42°C for 48 hours under micro- aerophilic conditions. After incubation, the liquid medium was aspirated and centrifuged and the supernatent containing the exotoxin was decanted and retained.

b. Toxin assay

Cytotoxin produced by some strains of C. jejuni is indicated by destruction of cells in culture systems.

Filtrates of C. jejuni cultures cause rounding of cells and death when incubated for 24-48 hours with CHO cell monolayers. Cell death was proportional to the concentration of toxin present. Toxin production is enhanced by culture of C. jejuni in a biphasic system comprising Mueller-Hinton medium as the solid phase and

Medium 199 as the liquid phase.

An aliquot of 0.1 ml of Eagle's Minimum Essential Medium (MEM) (Gibco Laboratories) containing approximately 5 X 103 CHO cells was added to each of the 96 wells of a flat bottomed culture plate. The plate was incubated at 37°C under 5% carbon dioxide for 18 to 24 hours to facilitate fixation of cells. Supernatant containing the exotoxin diluted in Medium 199 was then added to the wells in aliquots of 0.1 ml. The plate was incubated as before and the cells were monitored for 5 days. Cell death indicated the presence of exotoxin. Chapter 13

RESULTS

13:1. Prevalence studies

13:1(1). Prevalence in avian species

Submissions to the Louisiana Veterinary Medical

Diagnostic Laboratory included commercial chickens, backyard Galliformes and exotic and companion species as listed in Table 9.

Psittaciformes were most frequently submitted followed by Galliformes, Passeriformes and Anseriformes (Table 10).

Of a total of 445 cases examined, C. jejuni was

isolated from 45 cases (10.1%). The isolation rate was highest in August as shown in Figure 5. Birds of the order Galliformes yielded the highest isolation rate

(25.2%) followed by Anseriformes (12.9%) and Columbiformes (8.3%) (Table 10). The isolation rate from Falconiformes

102 Table 9. Species of birds examined.

ORDER FAMILY SPECIES NUMBER EXAMINED

Struthioniformes Struthionidae Ostrich (Struthio camelus) 1

Pelecaniformes Pelecanidae Brown Pelican (Pelecanus occidentalis) 2 Phalacrocoracidae cormorant (sp?) 1

Ciconiiformes Ciconiidae Saddle-billed Stork (Ephippiorhynchus asiaticus) 1

Scopidae Hammerhead Stork IScopus umbretta) 1

Threskiornithidae Sacred Ibis (Threskiornis threskiornis) I Scarlet Ibis (Eudocim ruber) 1 Roseate Spoonbill (Ajaia ajaja) 1

Anseriformes Anatidae domestic swan (Cygnus olor) 1 Black-necked Swan (Cygnus melanocoryphus) 1 Whooper Swan (Cygnus cygnus) 1 domestic goose (Anser anser) 1 Muscovy Duck (Cairina moschata) 2 Mood Duck (Aix sponsa) 1 Mandarin duck (Aix galericulata) 3 American Wigeon (Anas americana) 3 Gadwall (Anas strepera) 2 Common (Green-winged) Teal (Anas crecca carolinensis) 3 Mallard (Anas platyrhynchos) wild A domestic Mallard 3 domestic duck 5 domestic runner duck 1 Chinese Spotbill Duck (Anas poecilorhyncha} Northern Pintail (Anas a cuta) Northern Shoveler (Anas clypeata)

Falconiformes Cathartidae King Vulture (Sarcoratiphus papa)

Accipitridae Mississippi Kite (Ictinia nississippiensis) Bald Eagle {Haliaeetus leucocephalus} Broad-winged Hawk (Buteo platypterus) Red-tailed Hawk (Buteo jaoaicensis) hawk (Buteo sp.I Golden Eagle (Aquila chrysaetos) 104

Table 9 cont’d.

Galliformes Cracidae Crested Guan (Penelope purpurascensj 1

Heleagrididae Wild Turkey (Heleagris gallopavo] 8 domestic turkey T

Phasianidae Bobwhite Quail (Colinus virginianus) 32 Chukar Jector/s chukar) 1 Jungle Fowl (Callus gallus) 3 game fowl 44 domestic chicken 109 Ring-necked Pheasant (Phasianus colchicus) 6 Red Golden Pheasant (Chrysolophus pictus) 2 Gray Peacock Pheasant (Polyplectron bicalcaratum) 1 Common Peafowl (Pavo cristatus) 13

Grui formes Gruidae Sarus Crane (Grus anti gone) Demoiselle Crane (Anthropoides virgo)

Rallidae American Coot (Fulica atra)

Columbiformes Columbidae Rock Dove (Columba livia) Ring-necked Turtle Dove (Streptopelia risoria) domestic dove Mourning Dove IZenaida macroura)

Psittaciformes Loriidae Red Lory (Eos bornea) 1 Rainbow Lory (Trichoglossus haematodus) 1 Black-capped Lory (Lorius lory) 1 lory (sp?) 1

Cacatuidae Palm Cockatoo (Probosciger a t t e r i m ) 1 Salmon-crested (Holuccan) Cockatoo (Cacatua moluccensis) 4 White (Umbrella) Cockatoo (Cacatua a l b a ) 2 Coffin’s Cockatoo (Cacatua go f f ini) 2 cockatoo (sp?) 2 Cockatiel (Nysiphicus hollandicus) 45

Psittacidae Grand Eclectus Parrot lEclectus roratus roratus) 3 Red-sided Eclectus Parrot (Eclectus roratus polychloros) 4 Budgerigar (Helopsittacus undulatus) 57 African Grey Parrot (Psittacus eritbacus) 1 Senegal Parrot (Poicephalus senegalus) 1 Gray-headed (Madagascar) Lovebird lAgapornis cana ) 1 1 0 5

Table 9 cont’d.

Psittacidae Peach-faced Lovebird (Agapornis roseicollis) 4 cont’d. Masked Lovebird (Agapornis personata) 1 lovebird (Agapornis sp.) 5 Rose-ringed (Ring-necked) Parakeet {Psittacula krameri) 3 Green-winged Macaw (Ara chloroptera) 1 Chestnut-fronted (Severe) Macaw (Ara several 1 Red-shouldered (Noble) Macaw (Ara nobilis) 1 macaw (Ara sp.) 2 Mitred Conure (Aratinga mitrata) 1 White-eyed Conure (Aratinga leucophthalmus) 1 Jandaya (Jenday) Conure (Aratinga jandaya) 1 Black-hooded (Nanday) Conure (Nandayus nanday) 3 conure (sp?) 2 Honk (Quaker) Parakeet (Hyiopsitta monachus) 2 White-fronted (Spectacled) Amazon (Amazona albifrons) 2 Red-lored Amazon (Amazona autumnalis) 3 Blue-fronted Amazon (Amazona aestiva) 3 Yellow-crowned Amazon (Amazona ochrocephaJa) 2 Yellow-naped Amazon (Amazona ochrocephala auropalliata) 2 Double Yellow-headed Amazon (Amazona ochrocephala oratrix) 5 Orange-winged Amazon (Amazona amazonica) 1 Blue-crowned Mealy Parrot (Amazona farinosa guatemalae) 1 Amazon Parrot (Amazona sp.) 5 parrot (sp?) 2

Musophagiformes Husophagidae Guinea Turaco (Tauraco persa) 2

Strigiformes Strigidae Eastern Screech Owl (Otus a s i o) 1 Great Horned Owl (Bubo virginianus) 1 Barred Owl (Strix varia) 4 owl (unknown) 1

Piciformes Rhamphastidae toucan sp. (unknown) 1

Passeriformes Fringi11idae Common Canary (Serinus canaria) 22 Yellow-fronted Canary (Serinus mozambicus) 2 House Sparrow (Passer donesticus) 1

Estrildidae Zebra Finch (Poephila guttata) 3

unknown finch (unknown) 4 1 0 6

Table 9 cont’d.

Sturnidae Common Starling (Sturnus vulgaris) 1

Corvidae Blue Jay (Cyanocitta cristata) 1 Table 10. Isolation of C . jejuni from different orders of avian species

Submissions C. jejuni isolation

a Orde rs # % # "O

Psittaciformes 179 40 . 2 1 0.6

Galliformes 151 33.9 38 25.2

Passeriformes 36 8 .1 0 0-0 Anse ri formes 31 7 . 0 4 12.9 Falconi fo rmes 13 2.9 1 7.7 Columbi formes 12 2 . 7 1 8 . 3

Strigiformes 7 1.6 0 0.0

Ciconiiformes 5 1.1 0 0.0

Grui formes 4 0.9 0 0.0 Pelecaniformes 3 0.7 0 0.0

Musophagi formes 2 0.5 0 0 . 0

Pici formes 1 0 . 2 0 0.0

Struthioni formes 1 0.2 0 0.0

Total 445 100.0 45 10.1 X iue : esnl aito i ioain f . jejuni C. of isolation in variation Seasonal 5: Figure m n as m r x ra w‘tj FEB-APR MAY-JUL rm va species. avian from PERIOD NOV-JAN FEB-APR MAY-JUL was 7.7% and less than 1% from Psittaciformes.

Campylobacter jejuni was not isolated from Passeriformes nor from other miscellaneous birds which were examined. Despite the larger number of submissions from the order Psittaciformes compared to Galliformes, the isolation rate

from the companion Psittacines was only 0.6% compared to 25.2% from the various types of poultry.

A total of 93 Anseriformes, including specimens derived from wildlife sanctuaries, were examined during the period of the study (Table 11). Campylobacter jejuni was isolated from 5 of the waterfowl yielding an isolation rate of 5.3%.

Death was attributed to various etiologic factors in cases from which C. jejuni was isolated. No mortality could be ascribed to campylobacteriosis (Table 12). Cases with endoparasitism as the diagnosed cause of death yielded the highest (64.7%) isolation rate of C. jejuni followed by cases with fungal infection (24.1). Other cases yielded relatively lower isolation rates. Table 11. Isolation of C . jejuni from waterfowl

Species Tested C. jejuni +ve

Duck (Anas platyrhynchos) 29 4

Teal (Anas crecca carolinensis) 28 0

Gadwall (Anas strepera) 22 0

Pintail (Anas acuta) 6 0

Wigeon (Anas americana) 3 1

Shoveller (Anas clypeata) 1 0

Goose (Anser anser) 1 0

Swan (Cygnus olor) 2 0

Total 93 5 Table 12. Etiological diagnosis in avian species

yielding C. jejuni

Disease condition C. jejuni +ve/ %

# examined

Endoparasitism 11/17 64.7 Fungal infection 7/29 24.1 Protozoan infection 3/19 15.8

Toxicity 2/18 11.1

Trauma 2/21 9.5

Nutritional deficiency

and starvation 2/25 8.0

Viral infection 4/59 6.8

Bacterial infection 7/113 6.2

Others (Undiagnosed) 7/14 4 4.8

Total 45/445 13:1(2). Prevalence in humans

Fecal specimens from 1423 human diarrheic patients were examined for C. jejuni during the period August 1987 through July 1988. Clinical symptoms in these patients ranged from watery diarrhea to more severe enteritis with bloody, mucoid stools, abdominal pain and pyrexia. Campylobacter jejuni was isolated from 38 patients yielding a prevalence rate of 2.7%. Of these isolates, 3 could not be propogated subsequent to initial isolation and identification.

A seasonal fluctuation in prevalence of campylobacter- iosis was noted with a sharp increase in frequency of isolation during the summer of 1988 and a smaller peak during the winter of 1987 (Figure 6 ). Monthly breakdown of the number of fecal samples examined and the number yielding C. jejuni and Salmonella spp. during the period of study are depicted in Table 13.

The age of patients ranged from 2 to 50 years. A high prevalence was observed in children under 5 years, and in adults aged 21 to 25 and 31 to 40 years (Figure 7). Both iue : esnl aito i ioain f C^_ of jejuni isolation in variation Seasonal 6: Figure X AUG-OCT mnssmrxrawu SPECIES ♦ ♦ » C. JEJUNI B-B-B SALMONELLA SP. SALMONELLA B-B-B JEJUNI C. » ♦ ♦ SPECIES n Sloel sp fo ua patients. human from spp. Salmonella and NOV-JAN PERIOD FEB-APR MAY-JUL 113 Table 13 : Isolation of C. jejuni and Salmonella spp.

from human diarrheic patients

Month # of fecals C. jejuni Salmonella spp.

examined +ve % +ve %

87 Aug 109 1 0.9 7 6.4

Sep 102 3 2.9 4 3.9

Oct 97 2 2.1 2 2.1

Nov 108 3 2.8 1 0.9

Dec 105 5 4.8 1 1.0

88 Jan 129 2 1.6 4 3.1

Feb 126 2 1.6 2 1.6

Mar 148 2 1. 4 1 0.7

Apr 109 1 0.9 2 1.8

May 141 3 2.1 2 1.4

Jun 111 10 9.0 2 1.8

Jul 138 4 2.9 1 0.7

Total 1423 28 2.7 29 2.0 PERCENTAGE 30

AGE GROUP (in years)

Figure 7. Age distribution of human patients with campylobacteriosis. 116 sexes were evenly represented in the sample (52% males,

48% females).

Prevalence of Salmonella spp. during the study period was 2.0%. A peak occured in fall of 1987 and the isolation rate declined thereafter. 1 3 :2 . Serotyping

Antibody titers of antisera prepared against 16 reference strains of C. jejuni are depicted in Table 14.

Except for antiserum 25, all had titers of 1:320 and above.

Table 15 depicts the serotype distribution among human and avian isolates.

Serotypes 4, 5 and 10 were most prevalent among the isolates from humans, while serotypes 1, 4 and 16 were most common among avian isolates. Serotypes 2, 3, 4, 5,

10, 16, 34 and 36 were common among both human and avian isolates, of which serotypes 2, 4, 5 and 16, were isolated in decreasing frequency.

Serotypes 18 and 28 were obtained only from humans while serotypes 1, 21 and 49 were isolated from avian species. Serotypes 25, 37 and 56 were not identified in either humans, or avian species. Figure 8 compares the frequency of isolation of serotypes of C. jejuni from humans and avian species. Table 14 : Homologous and heterologous reactions of C. jejuni serotyping antisera

Titers of serotyping antisera against C .jejuni reference strains

Ref. 1 2 3 4 5 10 16 18 21 25 28 34 36 37 49 56 str .

1 320 ------2 - 640 ------3 __ 640 ------4 _ _ _ 640 ------5 _ 1280 ------10 _____ 1280 ------16 ______320 ------18 ______640 ------21 ______640 ------25 ______>160 ------28 ______320 ----- 34 ______640 ---- 36 - >320 - - - 37 ______640 -- 49 - >320 - 56 ______640 Table 15. Serotypes of C. jejuni isolated from human

patients and avian species

Prevalence in Total

Serotype Human Avian Prevalence

# % # % # %

1 0 0.0 10 22.2 10 13.3

2 2 6.7 5 11.1 7 9.3

3 2 6.7 1 2.2 3 4.0

4 5 16.7 6 13.3 11 14.7

5 6 20.0 3 6.7 9 12.0

10 6 2 0 . 0 1 2.2 7 9.3

16 3 10.0 6 13 . 3 9 12.0

18 2 6.7 0 0.0 2 2.7

21 0 0.0 2 4.5 2 2.7

28 1 3.3 0 0.0 1 1.3

34 1 3.3 2 4.5 3 4.0

36 1 3.3 4 8.9 5 6.7

49 0 0.0 1 2.2 1 1.3

UT 1 3.3 4 8.9 5 6.7

Total 30 100.0 45 100.0 75 100.0

UT : Untypable Avian Human

Serotype

22.2% 1 | 0 .0 %

1 1 .1 % 2 | 6.7%

2 .2% 3 1 6.7%

13.3% 4 1 16

6.7% 5 1 20%

2 .2 % 10 | 20%

13 . 3% 16 | 10%

0 .0 % 18 | 6.7%

4.5% 2 1 1 0 .0 %

0 .0 % 28 | 3 . 3%

4 . 5% 34 | 3 .3%

8.9% 36 | 3 . 3%

2 .2% __ 49 | 0 .0 %

8.9% UT | 3 .3%

UT : Untypable

Figure 8 . Comparison of C. jejuni serotypes isolated

from human patients and avian species Among the avian isolates, 60% belonged to serotypes 1,

2, 4 and 16. Serotypes 4, 5, 10 and 16 constituted 67% of human isolates.

13:3. Toxigenicity study

Each determination of GMl ELISA titer represented a mean of 9 wells and the procedure was repeated twice to minimize experimental error. Similarly each CHO cell assay represented the mean of counts from 4 wells and was repeated twice to ensure consistency. The sensitivity of

GMl ELISA and CHO cell assay was comparable and the results were complimentary in determining the toxigenicity of C. jejuni isolates.

Enterotoxin production was detected in 53% of human and

49% of avian isolates. Cytotoxin production was detected in 49% of human and 62% of avian isolates (Table 16).

Both enterotoxin and cytotoxin were produced by 28% of human and 29% of avian isolates. Neither factor was Table 16. Enterotoxin and cytotoxin produced by C. jejuni

isolates derived from either human patients or

avian species

Type of toxin # of isolates Toxin +ve %

Avian Enterotoxin 45 22 49

Cytotoxin 45 28 62

Human

Enterotoxin 87 46 53 Cytotoxin 87 43 49 detected in 25% of human and 18% of avian isolates respectively (Figures 9 and 10).

This study showed that 75% of human and 82% of avian isolates produced either or both toxins while 25% and 18% respectively were non-toxigenic.

Toxin production by the 5 most frequently isolated serotypes were compared. Eighty seven percent of isolates of serotype 5 produced enterotoxin, 64% of serotype 1 produced cytotoxin and 64% of serotype 10 produced both toxins (Figure 11 and 12).

Production of either toxin was detected in 86 % of serotypes 4 and 5, 82 % of serotype 10, and 79 % of serotype 1 . 124

Figure 9 : Distribution of toxin-producing C. jejuni isolates derived from human patients

C T N + v e C T N -ve

1

C T X + v e 28% | 2 2 %

1

1

C T X - ve 25% | 25%

1

CTX: cytotoxin; CTN: enterotoxin Figure 10: Distribution of toxin-producing C. jejuni

isolates derived from avian species

CTN +ve CTN -ve

1 CTX +ve 29% | 33%

1

1

CTX -ve 2 0 % | 18%

!

CTX: cytotoxin; CTN: enterotoxin 126

FREQUENCY 15

O 10 16 18 21 25 78 3'* 36 37 /+9 56 SEROTYPE

CTX □ NEGATIVE [*5533 POSITIVE

Figure 11. Cytotoxin production by C. jejuni isolates from both avian species and human patients. 15

14

13

12

1 1

10

9

8

7

6

5

4

3 $ 2 .*y

1 & &

0 1 2 3 4 5 10 16 18 21 25 20 34 36 37 49 50,

SEROTYPE CTN I "I NEGATIVE CXXXX) POSITIVE

Enterotoxin production by C^_ jejuni isolates from both avian species and human patients. Chapter 14

DISCUSSION

14:1. Prevalence in avian species

The present study shows the significance of Galliformes as a reservoir of C. jejuni. The prevalence rate of

25.2% is significant compared to other orders. It is noted however that this study was limited to traumatized or diseased birds submitted for necropsy. The assumption that the data obtained represents the prevalence of

C . jejuni in the population of commercial, companion and free living avian species in Louisiana, is constrained by the degree of correspondence between the sample and the population of birds in the state.

Although Psittacines were submitted for examination in relatively large numbers, a prevalence of only 0 .6 % was recorded. This suggests that Psittacines do not represent a risk of transmission of C. jejuni to human contacts.

Due to relative isolation and cage housing of these birds, an infectious cycle cannot be maintained and C. jejuni infection in individual birds is rare. The susceptibility

128 129 of Psittacines to C. jejuni infection in aviaries, breeding colonies and quarantine stations should however be investigated.

A 12.9% isolation rate from Anseriformes indicates the potential for these birds to transmit infection.

Migratory waterfowl can serve as asymptomatic carriers of infection over long distances. It would be of interest to study the prevalence of C. jejuni in various migratory and non-migratory species and in water.

In the series of birds reviewed, mortality was not attributed to C. jejuni infection. Although statistical analysis is inappropriate due to biased sampling, the results suggest a positive correlation between parasitism and C. jejuni isolation. Fungal infections too, show a similar positive correlation. Higher isolation rates are presumably due to increased colonization of C. jejuni in the intestinal tract following alteration in immune status attributable to parasitic and fungal infections. Further investigation is necessary to substantiate this observation.

Backyard Galliformes and other orders function as healthy carriers and may represent a potential zoonotic risk to owners following contamination of water, the environment, home-dressed poultry and eggs. 14:2. Prevalence in humans

The prevalence rate in diarrheic human patients in Baton Rouge (2.7%) corresponds to the 2.6% prevalence rate recorded in a Texas (Subramanyam et al., 1984). The

results of this survey represent a basis for subsequent epidemiological investigations. It is noted that the sampled population is biased since it comprised diarrheic patients. The results are not intended to represent the prevalence of C . jejuni in the human population of Baton

Rouge. The information obtained may certainly be used to characterize the population of patients with diarrhea and gastrointestinal symptoms sufficient to justify professional intervention. The socio-economic and demographic factors relating to the sampled population may differ from patients seeking treatment at charity hospitals. The actual prevalence rate for the Louisiana population will obviously be lower than the value obtained in this study due to the selection of diarheic individuals.

Seasonal fluctuation in prevalence was demonstrated in the present study. An increase during summer may be attributed to warmer temperature but indirect social factors may include seasonal frequency of picnics and barbeques, which are associated with food-borne infection.

Similar findings were documented in a study conducted by

Tauxe et al. (1987). The second peak of campylo- bacteriosis, demonstrated in the present study, occured during December, which may correspond to the November peak in the national study conducted by Tauxe et al. (1987).

The winter peak is probably due to social interaction and changes in food consumption during November and December coinciding with Thanksgiving and Christmas.

High isolation rates recorded in infants is in all probability due to perinatal transmission and has been documented by other workers (Karmali et al., 1984; Young et al., 1985). The increase in prevalence rate noted in young adults is attributed to lifestyle. Consumption of larger quantities of poultry, red meat and seafood increases the risk of food-borne infection. Recreational and occupational exposure which brings humans into contact with animals or animal products is an important factor contributing to the higher rate of isolation in the 20 to

40 year age group. 14:3. Serotyping

The Penner system was able to type 91.1% of the isolates obtained from human patients and 96.7% of avian isolates using antisera prepared against 16 out of 60 known reference strains. The 16 serotypes selected had previously been shown to occur most frequently in humans and animals in North America (Penner et al., 1983; Patton et al., 1985). It was not possible to serotype 8.9% of human and 3.3% of avian isolates respectively and this may be attributed to either isolates cooresponding to one of

44 other serotypes not included in this study or rough variants which do not conform to the Penner system.

Fourteen of the 16 serotypes were identified among the isolates collected in the study. Serotypes 2, 3, 4, 5, 10, 16, 34 and 36 were common to both avian species and humans. This indicates the potential for zoonotic transmission of C. jejuni infection from birds. Serotype

1 was isolated only from avian species indicating the low risk this serotype pose for human infection.

Correspondance was noted between the results in the present study and the data of Penner et al. ( 1983) and Patton et al. (1985) as depicted in Table 17. Comparing the frequency of isolation, it is seen that serotypes 2 ,

4, 5 and 16 are amongst the five most common in both the

CDC sample and this study. Serotype 10 is included among the five common isolates in this study but not in the CDC sample. Although serotype 1 was not isolated from human patients in the Baton Rouge sample, it is within the first five in the CDC list of isolates.

Seven out of 35 (20%) human and 8 out of 45 (18%) avian isolates respectively reacted to more than one antiserum. Antigens 4 and 16 were closely related, appearing in pairs 4 times. Antigens 2 and 10 were similarly related, appearing 3 times. Other combinations included 2(56) recorded twice, 3(21,34), 4(2), 10(18), 16(2) and 34(2) appearing only once.

14:4. Toxigenicity study

A relatively larger proportion of isolates belonging to serotypes 5 and 10 produced enterotoxin. Cytotoxin production was detected in 64% of both serotypes 1 and 10.

Consistent production of either toxin was detected in serotypes 1, 4, 5 and 10. Since these serotypes are Table 17 : Most common serotypes of C. jejuni isolated:

Three studies compared

% prevalence

Serotype Toronto1 Georgia2 Louisiana3

1 10.3 10.8 13.5

2 14.4 10.8 9.5

3 7.0 5.1 4.1 4 14.3 12.7 13.5

5 4.0 8.9 12.2

10 - 5.1 9.5

16 - 5.7 12.2

18 2.1 4 . 5 2.7

21 1.9 4.5 2.7

36 - — 6.7

1. Penner et al. (1983), University of Toronto, Canada. 2. Patton et al. (1985), Center for Disease Control,GA.

3. Present study, Louisiana State University. frequently isolated both from human patients and avian

species, there is considerable potential for human

infection with toxigenic strains of C. jejuni.

Toxin was produced by 75% of human and 82% of avian isolates. The relative percentages of isolates producing

toxins, is in conformity with the results of Johnson and

Lior (1986) who reported that 85% of human and 100% of

avian isolates were toxigenic. The higher values may be due to differences in assessing toxin production iji vitro. Morphological changes in 30% of cells was regarded as indicative of toxin production compared to a 50% threshold

in this study.

This study identified the potential for toxigenic

strains of C. jejuni to infect humans, especially through the medium of contaminated poultry.

14:5. General considerations

Since this prevalence study is the first to be undertaken in Louisiana, the data may be used as a basis

for further investigation of the epidemiology of the disease. The present study comprises diarrheic patients in a specific hospital. A detailed study including

different socio-economic segments of the population and

healthy individuals should be undertaken to obtain a more precise estimate of the prevalence of human infection. A more comprehensive prevalence study including larger

numbers of free living and clinically normal commercial

flocks would provide an indication of C. jejuni infection in avian species.

Serotyping and studies on toxigenicity were undertaken to classify C. jejuni isolates and to identify common

toxigenic serotypes. Procedures to speciate, serotype and to determine the toxigenic profile of C. jejuni isolates are pivotal to epidemiologic investigations.

Correlation between isolation of C. jejuni and con­

current bacterial, viral and parasitic infections should be investigated. This would indicate circumstances under which C. jejuni may cause clinical disease in humans.

Since 50% of the isolates examined in this study were common to both humans and avian species, there is an obvious potential for zoonotic transmission. As 75% of all isolates examined produce either enterotoxin or cytotoxin, infection of humans may lead to clinical campylobacteriosis. in avian species, C. jejuni is generally apathogenic but most broiler and turkey flocks carry one or more serotypes in the intestinal tract. Similarly asymptomatic carriers have been demonstrated in humans (Rajan and Mathan, 1982). It is therefore suggested that factors other than toxins are involved in the occurence of clinical disease (Johnson and Lior, 1986). Newell et al. (1985) has demonstrated the role of the flagellum in colonization of the intestinal mucosa. The extent and type of toxin production is a major determinant of pathogenisis. The specific conditions under which

C. jejuni produces cytotoxin and enterotoxin are not clearly understood. Since production of toxins jni vitro is variable, it is presumed that the complex intraluminal conditions existing in the intestinal tract may influence pathogenicity.

Isolates derived from water have been shown to be avirulent in mice compared to isolates derived from clinical cases (Newell et al., 1985). Kazmi (1984) demon­ strated an enhancement of virulence of C. jejuni following passage in mice. The mechanism underlying this change is not understood but is a reflection of either selection of a toxigenic population within the serotype or activation of the cellular mechanism for toxin production. A study conducted in India (Mathan et al., 1984) failed to demonstrate any difference in the occurence of enterotoxigenic strains among isolates of C. jejuni derived from diarrheic and clinically unaffected children.

In contrast, Klipstein et al. (1985) demonstrated an association between cytotoxin production and a clinical history of bloody diarrhea and between enterotoxin production and watery diarrhea.

The present study identified the most common serotypes prevalent in the samples derived from human patients and avian diagnostic submissions in Louisiana. Some serotypes were demonstrated to be common to both humans and avian species. In addition, the ability of some serotypes to produce toxins was confirmed. Further investigation is needed to define the relative importance of enterotoxins and cytotoxins in the pathogenesis of human disease. The genetic and environmental effects on production of enterotoxins, cytotoxins and virulence factors should be examined in relation to the susceptibility of the host and the development of immunity. An important consideration concerns the reasons why specific serotypes are innocuous in the avian reservoir host but are apparently toxigenic in humans. Subsequent investigations should be directed towards the following aspects of interaction of C. jejuni with host systems:

1. Factors influenzing toxin production, intestinal colonization and pathogenecity

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KANGASUNDARAM YOGASUNDRAM

BORN July 26, 1943 Sri Lanka

MARITAL STATUS Married, 2 children, 12 years and 2 years

NATIONALITY Sri Lankan

EDUCATION Primary: Colombo Hindu College, Ratmalana, Sri Lanka

Secondary: St. Johns College Jaffna, Sri Lanka

College: University of Ceylon Peradeniya, Sri Lanka

PROFESSIONAL DEGREES B.V.Sc. (=D.V.M.) University of Ceylon, Sri Lanka, December 1969

M.S. School of Veterinary Medicine, Louisiana State University, August 1 985

Ph.D. School of Veterinary Medicine, Louisiana State University, December 1988

PROFESSIONAL APPOINTMENTS

1 983-present Research Associate, School of Veterinary Medicine, Department of Epidemiology and Community Health, Louisiana State University

1980-1983 Instructor, Royal Veterinary Institute, Bhutan (United Nations Development Program Assignment)

1979-1980 Headquarters Veterinary Officer, Department of Animal Production and Health, Sri Lanka

1970-1979 Field Veterinary Officer, Department of Animal Production and Health, Sri Lanka

1970 (6 mos) Research Assistant, Smithsonian Institute, Sri Lanka

162 163

MEMBERSHIP American Association of Avian Pathologists

American Veterinary Medical Association

Ceylon Veterinary Council

AWARDS Gamma Sigma Delta, Louisiana Chapter

The Society of Phi Zeta, Tau Chapter

The Society of the Sigmi Xi, Louisiana Chapter

PUBLICATIONS Yogasundram, K. and S.M. Shane: The Viability of Campylobacter jejuni on refrigerated chicken drumsticks. Veterinary Research Communications, 1986, 10:479-486.

Shane, S.M., D.H. Gifford and K. Yogasundram: Campylobacter j ejuni contamination of table eggs. Veterinary Research Communications, 1986. 10:4087-492.

Yogasundram, K., S.M. Shane, R.M. Grodner, R.E. Smith and E.N. Lambremont: Decontamination of Campylobacter jejuni on chicken drumsticks using chemicals and irradiation. Veterinary Research Communications, 1987, 11:31-40

Sang, F.C., S.M. Shane, K. Yogasundram, H.V. Hagstad, and M.T. Kearney: Enhancement of virulence of Campylobacter j ejuni by passage in chicks. Avian Diseases. 1988. (In press)

PRESENTATIONS Shane, S.M. and K. Yogasundram: Methods of reducing Campylobacter jejuni contamination of poultry meat. Southern Poultry Science Society, Atlanta, Georgia, January, 1985.

Shane, S.M., D.H. Gifford and K. Yogasundram: Prevalence and pathology of selected diseases of quail. American Veterinary Medical Association, Las Vegas, July, 1 985.

Shane, S.M. and K. Yogasundram: The significance of campylobacteriosis in the poultry industry. 34th Western Poultry Disease Conference, University of California, Davis, 1986. Yogasundram, K. and S.M. Shane: Campylobacter jejuni in domesticated birds in Louisiana. Southern Conference on Avian Diseases, Baton Rouge, Louisiana, March 1988. EXPERIENCE

Teaching Instructor at Royal Veterinary Institute, Bhutan (U.N. assignment). Clinical and preventive medicine, herd and flock health programs, epidemiology, 1980-1983.

School of Veterinary Medicine, Louisiana State University. Course #VMED 5464, Teaching of avian necropsy techniques, diagnostic procedures, collection and submission of samples for histopathology, virology, microbiology and parasitology, etc., to final year DVM students.

Diagnostics Routine diagnosis of avian diseases at the Louisiana State Veterinary Medical Diagnostic Laboratory. Diagnosis of diseases in all species of birds including commercial, pet, and exotic. Advice to clients regarding prevention, treatment, and management.

Ten years of experience in large animal, small animal, and poultry practice, prevention programs, diagnosis and treatment of diseases, farm visits to check production fluctuations, and promotion of production of milk, meat, and eggs.

M.S. THESIS Survival and decontamination of Campylobacter jejuni on poultry meat. August 1985.

Ph.D. DISSERTATION Epidemiology of Campylobacter ieiuni. December 1988.

INTERESTS Diagnosis of avian diseases, avian and comparative pathology, public health and research in related fields. DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Kanagasundaram Yogasundram

Major Field: V e te r in a r y M ed ical S c ie n c e s

Title of Dissertation: Epidemiology of Campylobacter jejuni

Approved: Mi i/L. ijor Professor and Chairman

Dean of the Graduate Schoolj^^

EXAMINING COMMITTEE:

______^______j_

9—

Date of Examination:

August 26, 1988