A National Study of Gastrointestinal Parasites Infecting Dogs and Cats in Australia

Carlysle Sian Holyoake Bachelor of Science (Veterinary Biology) Murdoch University Bachelor of Veterinary Medicine and Surgery Murdoch University

Division of Health Sciences School of Veterinary and Biomedical Sciences Murdoch University Western Australia

This thesis is presented for the degree of Doctor of Philosophy of Murdoch University 2008

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I declare that this thesis is my own account of my research and contains as its main content work which has not been previously been submitted for a degree at any other tertiary educational institution.

…………………………...

Carlysle Sian Holyoake

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Abstract

Despite the popularity of companion animal ownership in Australia, recent and comprehensive information with regard to the prevalence, epidemiology and public health significance associated with gastrointestinal parasites of pet dogs and cats in

Australia is largely lacking. The primary aims of this study were to close this knowledge gap and to evaluate the veterinarian’s perception, awareness and knowledge of GI parasites in their locality, from a veterinary and public health stand- point. This included sourcing information with regard to commonly recommended deworming protocols. The awareness of pet owners regarding parasitic zoonoses and the degree of education provided to them by veterinarians was also determined.

A total of 1400 canine and 1063 feline faecal samples were collected from veterinary clinics and refuges from across Australia. The overall prevalence of gastrointestinal parasites in dogs and cats was 23.9% (CI 21.7-26.1) and 18.4% (CI 16.1-20.7), respectively. Overall Giardia duodenalis was the most prevalent parasite in dogs

(9.3%, CI 7.8-10.8) followed by hookworm (6.7%, CI 5.4-8.0). Isospora felis was the most prevalent parasite in cats (5.6%, CI 4.2-7.0), followed by Toxocara cati (3.2%,

CI 2.1-4.3).

A highly sensitive and species-specific PCR-RFLP technique was utilized to differentiate the various hookworm species which can infect dogs and cats directly from eggs in faeces. Ancylostoma ceylanicum was detected for the first time in

Australia in 10.9% of the dogs found positive for hookworm. This was a significant finding in terms of the zoonotic risk associated with this parasite.

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The zoonotic potential of Giardia and Cryptosporidium was investigated by genetically characterising isolates recovered from dogs and cats. All but one of the

Giardia isolates successfully genotyped were host specific, indicating a low zoonotic risk. It was hypothesized that the lack of zoonotic Giardia Assemblages was a consequence of there being a low prevalence of Giardia in the human population. The

Cryptosporidium recovered from dogs and cats was determined to be Cryptosporidium canis and Cryptosporidium felis respectively, a finding which supports growing evidence that Cryptosporidium in companion animals is of limited public health significance to healthy people.

Very few of the veterinarians surveyed in the study routinely discussed the zoonotic potential of pet parasites with clients. Most of the veterinarians recommended the regular prophylactic administration of anthelmintics throughout a pet’s life.

The low national prevalence of GI parasites reported is most likely a consequence of the widespread use of anthelmintics by pet owners. There is an over-reliance on anthelmintics by veterinarians to prevent and control parasites and their zoonotic risk.

This has resulted in veterinarians becoming complacent about educating pet owners about parasites. A combination of routinely screening faecal samples for parasites, strategic anthelmintic regimes and improved pet owner education is recommended for the control of GI parasites in pet dogs and cats in Australia.

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Publications

Scientific publications arising from this research (note: recent change of surname from Palmer to Holyoake)

PALMER , C. S., TRAUB, R. J., ROBERTSON, I. D., HOBBS, R. P., ELLIOT, A., WHILE, L., REES, R. & THOMPSON, R. C. (2007) The veterinary and public health significance of hookworm in dogs and cats in Australia and the status of A. ceylanicum . Veterinary Parasitology, 145 , 304-13.

PALMER , C. S., THOMPSON, R. C., TRAUB, R. J., REES, R. & ROBERTSON, I. D. (2008) National study of the gastrointestinal parasites of dogs and cats in Australia. Veterinary Parasitology, 151 , 181-90.

PALMER , C. S., TRAUB, R. J., ROBERTSON, I. D., DEVLIN, G., REES, R. & THOMPSON, R. C. (2008) Determining the zoonotic significance of Giardia and Cryptosporidium in Australian dogs and cats. Veterinary Parasitology, 154 , 142-7.

THOMPSON, R. C. A, PALMER , C. S. & O’HANDLEY, R. (2008) The public heath and clinical significance of Giarida and Cryptosporidium in domestic animals. Veterinary Journal , 177, 18-25.

Explanation concerning the inclusion of published papers in the text of this thesis and the contribution of co-authors to the published papers.

In all of the published papers included in the text of this dissertation I am the first author and I conducted all the work myself with the assistance of laboratory technicians and in collaboration with staff at Murdoch University, who have been included as co-authors on the papers.

Please note that as a consequence of this thesis being composed of published papers there is some repetition, particularly in the materials and methods section of each chapter.

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Conferences:

August 2007: oral presentation: Epidemiological investigation of intestinal parasites in domestic dogs and cats in Australia, International Congress of Parasitology, Glasgow, Scotland.

August 2007: poster: Nation wide survey of intestinal parasites in dogs and cats in Australia, International Conference of the World Association for the Advancement of Veterinary Parasitology, Gent, Belgium.

October 2005: oral presentation: Defining the relationships between pets, parasites and the people of Australia, International Conference of the World Association for the Advancement of Veterinary Parasitology, Christchurch, New Zealand.

September 2004: oral presentation: Risk Factors Associated with parasites of pets in Australia and Bangkok, Joint International Tropical Medicine Meeting Bangkok, Thailand.

July 2004: poster presentation: Risk factors associated with parasites of pets in Australia and Thailand, Australian Society for Parasitology annual conference, Perth, Australia.

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Acknowledgements

I would firstly like to convey my sincerest thanks to my three supervisors, Ian

Robertson, Andy Thompson and Rebecca Traub. Each always had an open door and offered unwavering encouragement, patience and guidance.

I am extremely grateful to Aileen Elliot and Gaby Devlin for their technical support with regard to processing samples and parasite identification.

I am particular indebted to Dr Bob Rees at Bayer Animal HealthCare, for not only supporting this project financially, but committing personnel to assist in the collection of samples. Without this assistance this project would not have been possible. In addition, I am grateful for the freedom and independence I was given to create and evolve this project.

I would like to thank Ryan O’Handley for encouraging me to think outside the square.

Lastly, without the belief, love and support of my husband Russell, my parent’s Cliff and Adele, and my brother Lincoln, this journey would not have been possible.

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Table of Contents

Abstract...... iii

Publications...... v

Acknowledgments...... vii

Table of Contents...... viii

List of Tables...... xiv

List of Figures...... xvi

1 Introduction...... 1

1.1 The need for this study...... 2

1.1.1 Companion animals in Australia...... 2

1.1.2 Public health significance...... 2

1.1.3 Drug resistance...... 3

1.1.4 The current situation...... 4

1.2 Previous studies conducted in Australia...... 6

1.2.1 Queensland...... 6

1.2.2 Australian Capital Territory...... 8

1.2.3 New South Whales...... 9

1.2.4 Victoria...... 11

1.2.5 Tasmania...... 13

1.2.6 South Australia...... 13

1.2.7 Western Australia...... 14

1.3 Common parasites and parasites of significance...... 19

1.3.1 Helminths...... 19

1.3.1.1 Hookworm...... 19

1.3.1.1.1 Infection in dogs and cats...... 19

1.3.1.1.2 Human infection...... 20

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1.3.1.2 Toxocara spp...... 22

1.3.1.2.1 Infection in dogs and cats...... 22

1.3.1.2.2 Toxocariasis...... 25

1.3.1.3 Trichuris vulpis ...... 26

1.3.1.4 Dipylidium caninum ...... 27

1.3.1.5 Spirometra erinacei ...... 28

1.3.1.6 Echinococcus granulosus ...... 29

1.3.1.6.1 Infection in dogs...... 29

1.3.1.6.2 Hydatid disease...... 31

1.3.2 Protozoa...... 33

1.3.2.1 Giardia duodenalis ...... 33

1.3.2.1.1 Dog and cat infection...... 33

1.3.2.1.2 Human infection...... 35

1.3.2.2 Enteric coccidians...... 37

1.3.2.2.1 Cryptosporidium ...... 37

1.3.2.2.2 Isospora ...... 39

1.3.2.2.3 Sarcocystis spp...... 40

1.3.2.2.4 Hammondia...... 41

1.3.2.2.4.1 Hammondia heydorni ...... 41

1.3.2.2.4.2 Hammondia hammondi ...... 42

1.3.2.2.5 Toxoplasma gondii ...... 42

1.3.2.2.5.1 Human infection...... 43

1.3.2.2.6 Neospora caninum ...... 44

1.4 Risk factors for parasitism...... 46

1.5 The importance of education in parasite control and the prevention of zoonotic disease...... 49

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1.6 The general aims of the study...... 52

2 General Materials and Methods...... 53

2.1 Study Area...... 54

2.2 Study Design...... 55

2.2.1 Overcoming the logistical problems of conducting a national study...... 55

2.2.2 Sample design...... 56

2.2.2.1 Stratification...... 56

2.2.2.2 Sample size...... 57

2.2.3 Education campaign ...... 59

2.2.4 The Use of Questionnaire Surveys...... 59

2.2.4.1 Questionnaire design and implementation...... 59

2.2.4.2 Pretrial of the Questionnaires...... 61

2.2.5 Study Implementation...... 61

2.2.6 Preservation and transportation of faecal samples...... 62

2.3 Parasitological techniques...... 62

3 National study of the gastrointestinal parasites of dogs and cats in Australia..64

3.1 Introduction...... 65

3.2 Materials and methods...... 71

3.3 Results...... 73

3.4 Discussion...... 80

4 The veterinary and public health significance of hookworm in dogs and cats in

Australia and the status of A. ceylanicum ...... 85

4.1 Introduction...... 86

4.2 Materials and Methods...... 89

4.2.1 Source of faeces...... 89

4.2.2 Parasitological procedures...... 90

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4.2.3 Molecular methods...... 91

4.2.3.1 DNA extraction...... 91

4.2.3.2 PCR for differentiating A. caninum, A. tubaeforme, A.

ceylanicum and Uncinaria stenocephala from A. braziliense ...... 91

4.2.3.3 PCR-linked restriction fragment length polymorphism

(RFLP)...... 92

4.2.3.4 Sequencing of A. ceylanicum positive samples...... 93

4.2.4 Morphological identification of specimens from the Queensland

Museum...... 94

4.2.5 Statistical analysis...... 94

4.3 Results...... 95

4.3.1 Prevalence of hookworm...... 95

4.3.2 PCR-RFLP...... 96

4.3.3 Morphological identification of specimens from the Queensland

Museum...... 100

4.3.4 Risk factors for parasitism...... 101

4.3.4.1 Multivariable analysis for dogs...... 101

4.3.4.2 Univariable analysis for dogs from veterinary clinics...... 102

4.3.4.3 Univariable analysis for cats...... 103

4.4 Discussion...... 104

5 Determining the zoonotic significance of Giardia and Cryptosporidium in

Australian dogs and cats...... 107

5.1 Introduction...... 108

5.2 Materials and methods...... 110

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5.2.1 Samples collected...... 110

5.2.2 Parasitological procedures...... 110

5.2.3 Molecular methods...... 110

5.2.3.1 DNA extraction...... 111

5.2.3.2 PCR amplification and sequencing of the Giardia

18SrDNA...... 111

5.2.3.3 PCR amplification and sequencing of the Giardia β-giardin

gene...... 112

5.2.3.4 PCR amplification and sequencing of the Crytosporidium actin

gene...... 113

5.2.4 Statistical analysis...... 113

5.3 Results...... 113

5.3.1 Microscopy...... 113

5.3.2 Risk factor analysis...... 114

5.3.3 Molecular characterization of Giardia and Cryptosporidium isolates..114

5.4 Discussion...... 115

6 Intestinal parasites of dogs and cats in Australia; the veterinarian’s perspective and pet owner awareness...... 119

6.1 Introduction...... 120

6.2 Materials and methods...... 121

6.3 Results...... 122

6.4 Discussion ...... 132

7. General Discussion...... 135

7.1 Gastrointestinal parasites in pet dogs and cats in Australia...... 136

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7.1.1 The need for more emphasis on education...... 137

7.2 Acknowledging the limitations of the current study...... 138

7.2.1 Dogs and cats sampled...... 138

7.2.2 Sampling...... 139

7.2.3 Methodology...... 140

7.2.4 Limitations in perspective...... 140

7.3 The power and conversely the limitations of using molecular tools ...... 141

7.4 Future research...... 143

7.4.1 Giardia ...... 143

7.4.2 Echinococcus granulosus ...... 144

7.5 Drug resistance...... 146

7. 6 Recommended approaches to control dog and cat GI parasites...... 148

Appendix 1 - Brochure for pet owners...... 150

Appendix 2 - Questionnaire for dog owners...... 156

Appendix 3 - Questionnaire for cat owners...... 165

Appendix 4 - Questionnaire for veterinarians...... 173

Appendix 5 - Refuge data sheet...... 183

References...... 184

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List of Tables

Table 1.1 Gastrointestinal helminths previously recorded in dogs and cats in

Australia...... 5

Table 1.2 Gastrointestinal protozoa previously recorded in dogs and cats in

Australia...... 6

Table 1.3 Assemblages of G. duodenalis (based on the allozymic analysis of

multilocus enzyme electrophoresis by Monis et al ., (1999); Monis et al .

(2003) and the subsequent species names proposed by Thompson and

Monis (2004))...... 36

Table 1.4 Risk factors associated with the presence of gastrointestinal parasites in

dogs and cats...... 47

Table 2.1 2005 Statistics of Dogs and Cats in Australia (‘000) (Hill, 2006)...... 54

Table 2.2 Location of the samples collected...... 58

Table 3.1 Results of previous intestinal parasite studies in domestic dogs in

Australia (Prevalence %)...... 67

Table 3.2 Results of previous intestinal parasite studies in domestic cats in

Australia (Prevalence %)...... 69

Table 3.3 The mean prevalence (%) of gastrointestinal parasites of dogs from

refuge and veterinary clinics in Australia...... 74

Table 3.4 The mean prevalence (%) of gastrointestinal parasites of cats from

refuge and veterinary clinics in Australia...... 75

Table 3.5 The results of the multiple logistic regression model for relationships

between dog host variables and parasitism...... 77

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Table 3.6 The results of the multiple logistic regression model for relationships

between host and management variables and parasitism for dogs from

veterinary clinics...... 78

Table 3.7 The results of the multiple logistic regression model for relationships

between cat host variables and parasitism...... 79

Table 3.8 The results of the multiple logistic regression model for relationships

between host and management variables and parasitism for cats from

veterinary clinics...... 80

Table 4.1 Previous prevalence studies in Australia ordered chronologically ...... 88

Table 4.2 Restriction profile...... 93

Table 4.3 Prevalence of hookworm in dogs according to the state in Australia...96

Table 4.4 Prevalence of hookworm in dogs according to climate...... 96

Table 4.5 Location of A. caninum, A. ceylanicum and U. stenocephala positive

samples in dogs...... 100

Table 4.6 Location of A. tubaeforme and A. caninum positive samples in cats.100

Table 4.7 The results of the multiple logistic regression model for the

relationships between the variables examined and hookworm infection

in dogs...... 102

Table 4.8 The results of univariate analysis for the variables likely to influence

hookworm infection in dog’s from veterinary clinics...... 103

Table 4.9 The results of univariate analysis for the variables likely to influence

hookworm infection in cats...... 104

Table 6.1 How much of a problem veterinarians consider the following parasites

in dogs in their practice area...... 123

Table 6.2 Veterinarians concern for the zoonotic hazard of the following

parasites in dogs in their practice area...... 124

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Table 6.3 Prevalence of hookworm and Giardia in dogs from veterinary clinics

and refuges in major cities and regional areas in Australia...... 125

Table 6.4 How much of a problem veterinarians consider the following parasites

in cats in their practice area...... 126

Table 6.5 Veterinarians concern for the zoonotic hazard of the following

parasites in cats in their practice area...... 127

Table 6.6 Veterinarians recommendations (n= 46) concerning the prophylactic

administration of anthelmintics for the control of GI parasites...... 128

Table 6.7 Frequency with which the veterinarians surveyed (n=46) discussed the

potential zoonotic hazards with their clients of the following

parasites...... 129

Table 6.8 Dog owner (n = 676) and cat owner (n = 420) awareness of

zoonoses...... 130

Table 6.9 Source of information for pet owners regarding zoonoses...... 131

Table 6.10 Anthelmintic history for dogs (n = 810) and cats (n = 572) from pet

owner questionnaire ...... 132

List of Figures

Figure 2.1 Location of samples collected in related to the population distribution

in Australia...... 58

Figure 4.1 PCR of the ITS of all of the canine and feline hookworm species...... 97

Figure 4.2 RFLP of the PCR product RTGHF1-RTABCR1 following digestion

with restriction endonuclease HinFI...... 98

Figure 4.3 RFLP of the PCR product of RTGHF1-RTABCR1 following digestion

with restriction endonuclease RSaI...... 99

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Figure 4.4 Lateral view of male bursa of A. ceylanicum from a cat from

Townsville, Australia, collected by GM Heydon in 1926...... 101

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Chapter 1

Introduction

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1.1 The need for this study

1.1.1 Companion animals in Australia

The level of pet ownership in Australia is high (Hill, 2006) and 91% of pet owners consider their pets integral members of the family unit (McHarg et al ., 1995).

Compared to non-pet owners, people who own pets typically visit the doctor less often and use less medication, have lower cholesterol and blood pressure, they recover more quickly from illness and surgery, they also tend to deal with stress better and are less likely to report feeling lonely (Anderson et al ., 1992; Headey, 2003; Cutt et al ., 2007).

It was estimated that these benefits resulted in a saving of $AUD3.86 billion of national health expenditure in 1999-2000 (Headey et al ., 2002).

Given the importance of pets in Australian society and the close proximity with which they share our lives it is surprising that despite the veterinary and public health significance of intestinal parasites of dogs and cats, very few data are available on their prevalence. Yet despite this, parasites represent a major source of income for the veterinary industry and globally, antiparasitic drugs accounted for 45% of veterinary pharmaceutical sales in 1998 (Zajac et al ., 2000).

1.1.2 Public health significance

The persons at highest risk of acquiring a zoonotic infection include small children, pregnant women, the elderly and the immunocompromised (Juckett, 1997). These groups are at higher risk partly because of behavioural characteristics and partly because of immunological reasons. The approximate number of HIV infected persons in Australia is 26,000 and increasing (McDonald, 2007). Physicians and veterinarians in Wisconsin, USA, were surveyed about the risk and methods of preventing zoonotic diseases in immunocompromised people (Grant and Olsen, 1999). It was found that

2 most of the physicians surveyed were not very comfortable about advising patients on the role of animals in the transmission of zoonotic agents and associated risks, moreover, physicians indicated that veterinarians should play an equal or greater role in advising clients about zoonotic disease (Grant and Olsen, 1999). Schantz (1994) speculated that veterinarians were reluctant to advise clients about parasites because they themselves were not confident with the subject matter.

Education is imperative to minimise zoonotic transmission of parasites; however, before an education campaign can be developed and implemented, data needs to be collected on the prevalence of parasites, pet owner knowledge of parasitic infections

(particularly their zoonotic significance) and the role veterinarians are currently playing in disseminating information.

1.1.3 Drug resistance

For the control of gastrointestinal parasites, it is believed that most Australian veterinarians advocate the prophylactic administration of anthelmintics at frequent intervals (Kopp et al ., 2007b) and there is evidence that Australian pet owners are diligent in treating their pets regularly for parasites (Schantz, 1991). Yet, there is increasing evidence to show that an intensive interval treatment regime is likely to produce considerable selection pressure for anthelmintic resistance (Kopp et al .,

2007b).

Not relying solely on anthelmintics for the control of parasites and employing a strategic approach to their administration will help avoid the development of drug resistance. In order to develop appropriate recommendations for strategic antiparasitic treatment of pet dogs and cats it is necessary to have recent, accurate and

3 comprehensive data on the prevalence of intestinal parasites in dogs and cats of different ages and from different geographical regions (Schantz, 1999).

1.1.4 The current situation

An Australian wide study has never been conducted before and there is no current data available on the nature and prevalence of enteric parasitic infections in dogs and cats.

The findings of previous studies are limited in their value because they generally involved small numbers of animals in a particular location and many were restricted to high risk groups (see Tables 3.1 and 3.2). Consequently, it is difficult to compare the prevalence data recorded in previous studies due to differences in the demographics of the animals sampled, differences in the sensitivity of diagnostic tests utilised and certain parasites may have been overlooked (there has been a gradual increase with time in the awareness of protozoan parasites). In the last 15 years there has also been an increase in the regular prophylactic treatment of pets with anthelmintics and this is likely to have affected the prevalence of helminths.

Based on studies conducted previously the diversity of gastrointestinal parasite species occurring in Australia can be determined (Tables 1.1 and 1.2).

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Table 1.1 Gastrointestinal helminths previously recorded in dogs and cats in

Australia

Cats Dogs

*Toxocara cati *Toxocara canis

Toxascaris leonina Toxascaris leonina

*Ancylostoma tubaeforme *Ancylostoma caninum

*Ancylostoma braziliense *Ancylostoma braziliense

*Ancylostoma caninum *Uncinaria stenocephala

*Uncinaria stenocephala *Trichuris vulpis

*Strongyloides stercoralis *Strongyloides stercoralis

Strongyloides felis Spirocerca lupi

*Taenia taeniaeformis *Echinococcus granulosus

*Taenia serialis Taenia hydatigena

*Dipylidium caninum Taenia ovis

*Spirometra erinacei Taenia pisiformis

Oncicola campanulatus *Taenia serialis

Capillaria spp. *Dipylidium caninum

*Spirometra erinacei

* Zoonotic

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Table 1.2 Gastrointestinal protozoa previously recorded in dogs and cats in

Australia

Cats Dogs

Isospora felis Isospora canis

Isospora rivolta Isospora ohioensis complex

*Giardia duodenalis *Giardia duodenalis

Hammondia hammondia Hammondia heydorni

*Toxoplasma gondii *Cryptosporidium spp.

*Cryptosporidium spp. *Sarcocystis spp.

Besnoitia spp. Neospora caninum

*Sarcocystis spp. *Entamoeba histolytica

*Blastocystis spp. *Balatidium coli

*Entamoeba histolytica *Blastocystis spp.

* Zoonotic

1.2 Previous parasite studies conducted in Australia

1.2.1 Queensland

Setasuban and Waddell (1973) examined the species of hookworm occurring in dogs and cats in Queensland. Three species of hookworms were found in the animals necropsied in Brisbane and Cairns during 1971-72. The majority of infections in dogs and cats from Brisbane were with A. caninum (72.0%) and A. tubaeforme (73%) respectively. Ancylostoma braziliense was recovered in Cairns from 3 of the 10 dogs and 8 of the 10 cats necropsied.

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A total of 400 cats from refuges in Brisbane were examined post-mortem during 1979

(Wilson-Hanson and Prescott, 1982). This study examined four groups of domestic cats based on age and looked at the prevalence of both endoparasites and ectoparasites. Toxocara cati was found to be the most common parasite (24.5%), followed by A. tubaeforme (19%) and D. caninum (19%). Spirometra erinacei was found in 8.5% of cats and was generally found in older animals and was most prevalent during winter and spring. The authors suggested the reason for the older distribution of infection with S. erinacei was because of the feeding and hunting habits of older cats. Isospora felis was the most common coccidian parasite recovered

(10.5%) and was most common in very young (6 to 8 week old), apparently healthy, kittens; while I. rivolta was found in only 2% of cats. Wilson-Hanson and Prescott

(1982) concluded that the age of the cat and the season were important factors that should be considered when studying gastrointestinal parasitism in cats.

In May 1987, a 12 month study was undertaken to identify the major parasites present in sheep dogs from 14 properties in the Charleville area of Queensland (Cornack and

O'Rourke, 1991). A total of 112 dogs were examined and faecal samples were collected repeatedly at two monthly intervals. Ancylostoma spp. were the most prevalent (20.1%) nematode eggs recorded. Toxocara , Toxascaris and Trichuris eggs occurred infrequently. Dogs younger than 2 years of age had a higher prevalence of nematode eggs than older animals (P<0.01). The mean prevalence of D. caninum was

4.1%, which did not vary significantly between properties, age groups, month of sampling or surprisingly with the level of flea infestation. Taeniid type eggs were found in only five faecal samples and these samples originated from dogs that were fed either uncooked sheep or macropod offal during the study period (Cornack and

O’Rourke, 1991).

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Duda et al . (1998) set out to determine the prevalence of Blastocystis in a group of 72 dogs and 52 cats housed at a refuge in the Brisbane region. Faecal samples were collected per rectum, wet mounts were prepared and the samples examined by light microscopy. Of the faecal samples examined 70.8% dog and 67.3% cat samples contained Blastocystis spp.. There was no association between the presence of the parasite and either the age or gender of the host. There was also no relationship between high numbers of Blastocystis spp. in the faeces and the presence of diarrhoea or other gastrointestinal signs, which is unlike the situation in humans (Duda et al .,

1998). The nuclear morphology of the Blastocystis spp. recovered from the dog and cat samples was not sufficiently distinctive to allow differentiation from B. hominis found in humans and hence the zoonotic potential remained unresolved.

1.2.2 Australian Capital Territory

Pavlov and Howell (1977) examined 100 cats from two different sources in Canberra for gastrointestinal parasites. Forty-two of the cats were captured at rubbish dumps around the city and were considered to be stray cats, while 58 domestic cats were obtained from a refuge. All 100 cats were necropsied and it was found that the majority of cats (65%) were infected with at least one helminth parasite. More stray cats were infected (85%) than were domestic cats (50%), with the most common parasite being D. caninum (29%) followed by T. cati (25%). Pavlov and Howell

(1977) concluded that the high prevalence of infection in the domestic cats was mainly due to the ingestion of a wide variety of intermediate hosts and hence, when combined with the high level of infection in the stray cats of the region it would be difficult to maintain domestic cats free from infection.

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1.2.3 New South Wales

During the period of 1973 to 1975, 464 dogs and 404 cats from pounds in the Sydney metropolitan area were necropsied and examined for intestinal helminths (Kelly and

Ng, 1975). Overall they found that the most common helminths in dogs were D. caninum (67.5%), T. vulpis (60.6 %), A. caninum (35.6%) and T. canis (35.1%).

Ancylostoma braziliense was present in one of the dogs examined. Toxocara cati

(57.2%) followed by D. caninum (39.4%) and A. tubaeforme (34.7%) were the most common parasites in the cats examined. Infection with Toxocara in both dogs and cats appeared significantly greater in those animals less than six months of age. The authors suspected that during this time the degree of environmental contamination in

Sydney, particularly with Toxocara spp., hookworms and T. vulpis , was widespread and as a result they emphasised the potential risk of infection to humans.

Dent and Kelly (1976) set out to determine the prevalence of cestode parasites in a sample of dogs from the Central Tablelands of New South Wales. The survey was conducted as a result of human hydatidosis continuing to be a significant public health problem and because of the increase in condemnation of sheep carcasses from T. ovis in the area. In their survey they examined the intestinal contents of 66 euthanased dogs from a pound (refuge) in Blayney, and 301 purged dogs from farms across the region. Taenia pisiformis was the most prevalent parasite (22.3%) and statistical analysis revealed a significantly higher frequency (P=0.01) of infection in male dogs than females. Echinococcus granulosus was detected in 4.7% of the purged dogs

(rural farm dogs). In contrast only 1.5% of the necropsied dogs (urban dogs), were positive for E. granulosus (Dent and Kelly, 1976).

9

Davies and Nicholas (1977) conducted a survey of helminths in dogs from the

Goodradigee Shire, New South Wales. A survey of hospital records in the shire in

1971 revealed that 7 new cases of human hydatid disease were treated by surgery out of a shire population of only 2,447 during a five year period (1969-1973) (Davies and

Nicholas, 1977). According to the authors the conditions in this area favoured the transmission of dog tapeworms. The shire was heavily stocked with sheep and cattle and enjoyed a mild climate allowing prolonged survival of tapeworm eggs on the pasture. In 1971 a campaign for hydatid control was introduced, which advocated the cessation of feeding offal to dogs, keeping dogs away from home killing areas and the disposal of carcasses found on properties. During their survey, Davies and Nicholas

(1977) found that of the 110 dogs purged, E. granulosus was present in 7.2%. They concluded that this was probably an underestimate of the true prevalence because a single purge may not always reveal E. granulosus in an infected dog. The frequent occurrence of T. hydatigena (13.6%) also reflected the common practice of feeding home-killed sheep to dogs. The survey demonstrated that little progress had been made in the control of E. granulosus in this region .

In 1981, Collins et al . (1983) collected 110 dog and 71 cat faecal samples from animals presented to the University of Sydney Veterinary Hospital and Clinic, and examined them for sporozoan parasites. Isospora felis was found to be the most common parasite in cats (4.2%), followed by I. rivolta (1.4) and Sarcocystis spp.

(1.4%). The most common protozoan parasites of the dogs sampled were Sarcocystis spp. (20.9%) followed by Isospora spp. (5.5%). Protozoa were found more frequently in those animals fed raw meat than in those fed only processed food. Due to the examination of only one faecal sample from each animal and intermittent oocyst and sporocyst shedding by adult hosts, these results were likely to be an underestimate of

10 the true prevalence of sporozoan infections in the cats and dogs in Sydney (Collins et al ., 1983).

Collins et al . (1987) investigated the prevalence of Giardia in 50 cats and 150 dogs from refuges in the Sydney metropolitan area by examining both faecal and duodenal contents and then comparing the results. Overall it was found that 8 of the cats and 50 of the dogs were infected with Giardia , however only 16 of the 50 dogs, and 5 of the 8 cats were identified as positive by faecal examination. The authors concluded that

Giardia was common in dogs and cats in Sydney, and warned that only examining one faecal sample from each animal could drastically underestimate the prevalence due to the intermittent shedding of this parasite.

1.2.4 Victoria

During 1968 to 1969, 792 dogs from farming properties across Victoria were purged with arecoline hydrobromide and the purged material examined for tapeworms

(Jackson and Arundel, 1971). Taenia pisiformis was found to be the most common cestode followed by D. caninum (17.4%), T. hydatigena (12.1%), and E. granulosus

(3.0%). It was not surprising to the authors that a high percentage of farm dogs from across Victoria were infected with cestodes as the feeding of sheep offal was common practice, and rabbits and hares were plentiful in the region (Jackson and Arundel,

1971).

In 1978, 734 unclaimed pound dogs throughout north-eastern Victoria were necropsied and examined for parasites (Blake and Overend, 1982). The most prevalent parasites in this survey were D. caninum (57%), T. vulpis (41%), T. canis (38%) and

U. stenocephala (26%). The survey on helminth parasites of urban Sydney dogs by

11

Kelly and Ng (1975) also showed D. caninum, T. vulpis and T. canis to be the most prevalent species, along with Ancylostoma caninum which replaces U. stenocephala in that area. Blake and Overend stated that a large proportion (68.5%) of the dogs examined in the survey were under two years of age, and hence may account, in part, for the high prevalence of T. canis . Sarcocystis spp . and I. ohioensis were the most common coccidian parasites with prevalences of 26.3% and 16.2% respectively.

The most recent survey of intestinal parasites of dogs in Victoria was conducted from

1991 to 1992 and focused on the Geelong and Melbourne areas (Johnston and Gasser,

1993). Faecal samples were collected from four different groups of animals (stray dogs, kennel dogs, park dogs and dogs presented to veterinary clinics) and examined for parasites. Similar to other surveys (Swan and Thompson, 1986; Savini et al ., 1993;

Bugg et al ., 1999; McGlade et al ., 2003a), this study demonstrated the significant difference between parasite burdens in animals sourced from different origins. For example, overall S arcocystis spp. was the most prevalent parasite however the prevalence varied greatly between stray, kennel, park and veterinary clinic derived samples (35.3%, 4.5%, 14% and 7.9% respectively). Unfortunately, no information was collected with regard to the different diets of these animals; however one may assume that many of the stray dogs had fed on raw meat at some stage. The first report of Cryptosporidium in dogs in Australia was also made in this survey. Johnston and

Gasser (1993) found that the overall prevalence of Cryptosporidium was 11%. A limitation of their study was the lack of information gathered in order to identify risk factors associated with parasitism. The only risk factor examined was the origin of the dogs and no statistical analysis of this data was conducted. The authors concluded that certain dog populations in southern Victoria were important reservoirs for parasites

12 and further investigation was needed into the epidemiology and public health significance of these parasites (Johnston and Gasser, 1993).

1.2.5 Tasmania

In the wake of a very successful hydatid eradication programme there was a marked decline in awareness of the potential dangers of zoonotic diseases harboured by dogs in Tasmania (Milstein and Goldsmid, 1995). Subsequently, Milstein and Goldsmid

(1995) set out to reassess the zoonotic potential of urban dogs in Hobart focusing on gastrointestinal parasites. Faecal samples were collected from 55 refuge and clinic dogs, and on examination Giardia was found to be the most prevalent parasite

(14.5%) followed by T. canis (10.9%). This study also found that 1.8% of dogs were infected with Cryptosporidium which was lower than that reported by Johnston and

Gasser (1994) in pound dogs (11%). It should be noted that both of these studies were most likely an underestimate of the true prevalence of Cryptosporidium , as they relied on using a modified Ziehl-Neelsen staining technique and light microscopy. This procedure is not sensitive enough for detecting light infections and the efficacy of the test relies heavily on the experience of the personnel performing it (Morgan et al .,

1996). Milstein and Goldsmid (1995), in light of their findings, emphasised that many of the dogs in Hobart were a reservoir of potential zoonotic infections.

1.2.6 South Australia

The only published report of the prevalence of helminths in dogs and cats in South

Australia was carried out during the period of 1979-1981 (Moore and O'Callaghan,

1985). During this period some 1614 dog and 376 cat faecal samples were submitted to the Department of Agriculture for both routine examination and from cases of suspected parasitism. Toxocara cati was found to be the most prevalent parasite in

13 cats (5.3%), followed by T. leonina (3.7%) and T. taeniaeformis (2.7%), the most prevalent parasites in dogs were T. vulpis (8.8%), T. canis (6.4%) and U. stenocephala

(4.0%). Given that the samples examined partially consisted of animals suspected of parasitism the prevalence of parasites was considerably low, however, the study concentrated on helminths and did not examine for protozoa and the worming history of the animals was also unknown.

1.2.7 Western Australia

Shaw et al . (1983) during the period 1978-1981, compared the prevalence of parasites in urban cats from a refuge in Perth to the prevalence of parasites in cats from rural areas south east of the Swan River. The small intestines of these cats were examined post-mortem and of the parasites found D. caninum was the most prevalent in urban cats (28.5%) followed by T. cati (12.2%) and T. taeniaeformis (8.8%), while

S. erinacei had the highest prevalence in rural cats (42.3%) followed by T. taeniaeformis (34.6%) and D. caninum (11.5%). These results demonstrated a significant difference in the prevalence and type of helminths in urban and rural cats.

With regard to the high prevalence of S. erinacei and T. taeniaeformis in the rural cats the authors suggested that this was indicative of their dependence on hunting prey for food, and for S. erinacei the presence of open and fresh water and marsh land in the rural area. A seasonal variation in the prevalence of D. caninum was also noted, with this parasite being more prevalent in May to June (autumn and early winter). It was suggested that this observation may be related to the influence of temperature and moisture on the biology of the flea or louse intermediate host. Faecal samples from 59 urban and 30 rural cats were also examined using a sucrose solution (specific gravity

1.15) flotation method and from these results the prevalence of Isospora spp. was

14 higher in the faeces of urban cats (20.3%) compared to rural cats (6.7%). A patent infection with T. gondii was also found in one urban cat.

In 1986 a survey was conducted in Perth which specifically investigated the prevalence of Giardia in dogs and cats (Swan and Thompson, 1986). Faecal samples were obtained from three different populations of dogs and cats: pets, refuge animals and animals from breeding kennels and catteries. All samples were examined using a zinc sulphate flotation technique and the overall prevalence of Giardia in cats and dogs was found to be 14% and 21% respectively. Swan and Thompson (1986) stated that the prevalences obtained were more likely underestimates of the actual level of infection, because in most cases only one faecal sample could be obtained from each animal. Cats in catteries had a higher prevalence than pets or those from the refuges and it was proposed that this was because the cattery animals were housed together in groups compared to the individual housing of the refuge and pet cats, hence facilitating the transmission of this parasite. The prevalence of Giardia was greater in dogs from the refuge and breeding kennels, and it was proposed that this was because of a high level of environmental contamination which provided a continuous source of infection to susceptible dogs in these locations. Other factors such as stress and a low plane of nutrition may also have contributed to a higher level of infection in refuge dogs (Swan and Thompson, 1986). Although there was no breed or sex disposition, the prevalence of Giardia was higher in younger animals and Swan and Thompson

(1986) speculated that the reason for such an occurrence was due to the development of acquired resistance with age.

Thompson et al . (1988), prompted by the finding of viable hydatid cysts from a western grey kangaroo (Macropus fuliginosus ) shot close to Perth, investigated the

15 prevalence of hydatid infection in kangaroos and feral pigs living close to Perth. The results showed that hydatid disease was common in western grey kangaroos and feral pigs in water catchment and forestry regions on the outskirts of Perth. During the course of the study, three domestic dogs used for pig hunting in the area of the survey were purged with arecoline acetarsol. One dog was found to harbour adult E. granulosus . The authors speculated that the source of infection for the kangaroos and pigs infected was most likely dogs belonging to hunters. Thompson et al. (1988) concluded that the public health significance of their findings was alarming, particularly in view of the resistant characteristics of the eggs and the large area over which they can be dispersed. Survival of eggs would also probably be enhanced by the regular watering of grassed areas during summer (Thompson et al ., 1988). Two main issues of concern were raised by this study, the first being the possible contamination of drinking water with E. granulosus in water catchment areas; and secondly, at the time raw, unprocessed, kangaroo meat was being sold locally as pet food and hence could potentially act as a source of infection for pet dogs (Thompson et al ., 1988).

Between 1990 and 1991, a survey of Western Australian dogs for Sarcocystis spp. and other intestinal parasites was conducted by Savini et al . (1993). During their survey

132 faecal samples were collected from pet and refuge dogs from Perth, as well as from farm dogs from the shire of Albany in the south-west of the state. Overall a lower prevalence of Giardia was detected in their study compared with the results of

Swan and Thompson (1986) (10.6% and 21.0% respectively). Refuge dogs and younger animals were found more likely to be infected. The prevalence of Sarcocystis was very high in refuge dogs (66.6%), but lower in the pet dogs (23.9%). Feeding raw meat was identified as a significant risk factor for infection; yet, the species the meat came from was not a significant factor. The overall prevalence of infection with

16

Sarcocystis and Isospora (31.3% and 2.3% respectively) was comparable with that found in Sydney (20.9% and 5.5% respectively) by Collins et al . (1983). The authors concluded that the high level of infection with Sarcocystis in dogs demonstrated that they must be considered a significant potential reservoir of Sarcocystis infection to intermediate hosts in WA. Savini et al. (1993) found that, unlike the situation in other capital cities of Australia (Brisbane and Sydney), the level of infection with hookworm was very low, in fact none of the urban dogs examined were found to have hookworm. It was suggested that the sandy environment of Perth may account for these contrasting results. Unsporulated oocysts were noticed in the faeces of one dog and were presumed to be those of Hammondia heydorni (Savini et al ., 1993).

In comparison to the parasites Shaw et al. (1983) found in cats in the Perth region, the results obtained by Sargent (1997) differed markedly. Sargent (1997) found that protozoan parasites, particularly coccidians, were most common, with I. felis being the most prevalent parasite in refuge and clinic cats (12.9%), followed by I. rivolta

(6.1%), while T. cati was only found in 1.2% of cats. In addition, Cryptosporidium was also detected in two cats for the first time in Australia and genetic analysis revealed that the isolates found were different to isolates of Cryptosporidium derived from humans, suggesting that they may not be of zoonotic significance (Sargent et al .,

1998) and were probably the species now designated C. felis .

During 1996, canine faecal samples were collected from five sources in metropolitan

Perth (Bugg et al ., 1999). The prevalence of gastrointestinal parasitism was higher in pet shop puppies (51%), than in dogs from refuges (37%), breeding kennels (32.7%), veterinary clinics (15.6%) and exercise areas (5.3%). Protozoa, in particular Giardia , were detected more frequently (22.1%) than were helminths. After adjusting for other

17 factors, it was concluded that puppies less than 6 months of age, dogs living in households with more than one dog, and dogs from refuges were significantly more likely to be parasitised (Bugg et al ., 1999).

The most recent survey of gastrointestinal parasites of cats was carried out in Perth during 2001 (McGlade et al ., 2003a). A total of 418 faecal samples were collected from cats at four different locations: refuges, pet shops, breeding establishments and private households. All samples were examined for parasites using sodium nitrate flotation, zinc sulphate flotation and malachite green staining followed by microscopy.

Overall the level of parasitism detected by microscopy was very low with I. felis being the most prevalent parasite (4.5%) followed by T. leonina (2.2%) and I. rivolta

(1.4%). Forty of the 418 samples were randomly selected and screened using a polymerase chain reaction (PCR) procedure in order to detect the presence of any

Giardia or Cryptosporidium that may not have been detected by microscopy. Of the forty samples screened by PCR, Giardia was detected in 80% and Cryptosporidium was detected in 10%. Fourteen of the 32 isolates which were positive for Giardia were sequenced at the 18S-rDNA gene. All but one isolate most closely resembled G. duodenalis dog genotype Group 4 (which is the equivalent of Assemblage D)

(McGlade et al ., 2003b). Two of the four isolates of Cryptosporidium were sequenced and found to closely resemble C. baileyi and C. muris . McGlade (2001) suggested that the presence of these two species of Cryptosporidium were likely to be due to mechanical transmission after the ingestion of an infected wild bird ( C. baileyi ) or an infected mouse ( C. muris ). McGlade et al . (2003a) also examined the risk factors associated with parasitism and found that kittens less than six months of age, and cats living in households with more than one cat or with a dog were significantly more likely to be parasitised.

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1.3 Common parasites and parasites of significance

1.3.1 Helminths

1.3.1.1 Hookworm

1.3.1.1.1 Infection in dogs and cats

Optimum development of hookworm larvae takes place in warm, moist, sandy soil that is sheltered from direct sunlight. For this reason most cases of hookworm disease in dogs and cats occur during late spring, summer, and early autumn in temperate climates, particularly when mild weather is accompanied by adequate rainfall

(Bowman et al ., 2003). In optimal conditions transition from egg to third stage infective larvae can be completed within a week (Prescott, 1984). Large numbers of infective larvae have been associated with carelessly managed kennels and pet shops where faeces are allowed to accumulate. Unpaved runs are also particularly favourable for the perpetuation of hookworm because the faeces can mix with the soil (Bowman et al ., 2003).

The severity of hookworm disease in dogs and cats not only depends on the magnitude of the challenge but also the resistance of the host (Bowman et al ., 2003). Host resistance to infection depends on the age of the animal, acquired immunity and the ability of the individual to compensate for blood loss caused by hookworm. As dogs grow older, they become more resistant to hookworms whether or not they experience infection. The ability for an animal to compensate for blood loss is influenced by their haematopoietic capacity, nutritional status and by the presence or absence of other stressors (Bowman et al ., 2003).

There are two main modes of infection for dogs and cats; firstly through skin penetration of the infective larvae and secondly via the ingestion of larvae either from

19 the environment or a paratenic host (Bowman et al ., 2003). Larvae which penetrate the skin enter the dermis, where they are transported through the lymphatic vessels and veins to the lungs. In the lungs, they penetrate the alveoli and migrate through the lungs, up the trachea and down the oesophagus and into the intestines where they mature. In dogs more than three months old, A. caninum larvae may fail to complete the migration through the lungs and are arrested in the tissues, where they survive as dormant (hypobiotic) larvae. These larvae are capable of moving to the mammary glands during lactation and puppies then become infected through the milk (Bowman et al ., 2003). In contrast the larvae acquired through ingestion do not undergo tracheal migration; instead they develop for a period of time in the gastrointestinal wall before re-appearing in the lumen and maturing to adult worms (Bowman et al ., 2002).

The principal veterinary importance of hookworm arises from their ability to suck blood in their primary host (Traub et al ., 2004b). Ancylostoma caninum and A. tubaeforme cause the greatest blood loss, while U. stenocephala , A. braziliense and A. ceylanicum are not heavy blood feeders. Infection with A. caninum and A. tubaeforme usually manifests as peracute or acute haemorrhagic diarrhoea with accompanying dehydration and anaemia, and in puppies and kittens, severe infestations may result in death. Hookworm may also cause protein and fluid losses and malabsorption, which often results in decreased growth and performance of infected animals (Dunsmore and

Shaw, 1990).

1.3.1.1.2 Human infection

There are three known clinical syndromes which manifest from human infection: cutaneous larva migrans, classical hookworm disease and eosinophilic enteritis

20

(Schantz, 1991; Schad, 1994). Cutaneous larva migrans is the most common syndrome in humans (Schantz, 1991).

Cutaneous larva migrans

Ancylostoma braziliense is responsible for most cases of cutaneous larva migrans, while A. caninum and U. stenocephala are less often involved, and only rare cases have been reported from infection with A. tubaeforme or A. ceylanicum (Davies et al .,

1993). Infections are most commonly seen in people who have greatest exposure to contaminated soil such as electricians, plumbers, and other persons who have to crawl beneath a house or building, and in children and sun bathers who recline on wet sand

(Schantz, 1991). Most of the lesions are seen on the legs, buttocks and hands, but they can be found on any part of the body that has been exposed to soil. Migration of the slow-moving larvae in tunnels in the skin results in an allergic reaction. Ordinarily the lesions are self-limiting and the intense pruritis subsides (Schantz, 1991).

Classical hookworm disease

Although classical hookworm disease is usually caused by human hookworms, the zoonotic species A. ceylanicum can also cause this syndrome via percutaneous entry and migration to the intestines (Schad, 1994). Infections with A. ceylanicum tend to be milder than those caused by human hookworms. Typically with a light infection the person is asymptomatic but when the burden is heavier infected individuals will be anaemic, and may demonstrate clinical signs similar to those caused by the human hookworms (Yoshida et al ., 1971; Carroll and Grove, 1986).

21

Eosinophilic enteritis

Prociv and Croese (1990) reported an epidemic of eosinophilic enteritis (93 cases) in

Townsville, northern Queensland, Australia. They reported that eosinophilic enteritis was caused by the zoonotic hookworm A. caninum . The patients experienced severe abdominal pain, diarrhoea, weight loss, and melaena. The observed syndrome was interpreted as a local allergic reaction to the feeding hookworms. The findings by

Landmann and Prociv (2003) suggest that ingestion of A. caninum larvae is the most significant route of human infection leading to eosinophilic enteritis. They suggested that possible routes of oral infection were by drinking soil-contaminated water, eating soil-contaminated food (e.g. on fresh vegetables), or by eating infected meat.

Although not confirmed, Landmann and Prociv (2003) suggested that many grazing animals and free range poultry may become infected from soil contaminated by dogs.

Hence ingestion of meat from these putative paratenic hosts, if undercooked, might lead to larvae developing directly into adult worms in the human intestine.

1.3.1.2 Toxocara spp.

1.3.1.2.1 Infection in dogs and cats

The clinical symptoms of infection with Toxocara depend on the age of the animal and on the number, location, and stage of development of the worms (Overgaauw,

1997b). Infection with this parasite is highest in puppies and kittens up to 6 months of age (Scothorn et al ., 1965; Visco et al ., 1977; Visco et al ., 1978).

Scothorn et al . (1965) found that prenatal infection of puppies with T. canis was responsible for stillbirths and early puppy deaths; however this has not been reported in other studies. After birth, puppies can suffer a number of ailments associated with either the migrating larvae or adult worms in the intestine. Pneumonia may manifest

22 from the tracheal migration of the larvae, and puppies have been known to die within two to three days as a result (Overgaauw, 1997b). At an age of two to three weeks, puppies can show emaciation and digestive disturbances caused by mature worms in the stomach and intestine (Overgaauw, 1997b).

Infection with T. cati in kittens, in contrast to the situation in puppies, is primarily via the transmammary route. Larvae transmitted via the colostrum or milk undergoes development in the kitten without tracheal migration (Overgaauw, 1997b). Thus in comparison to puppies, kittens tend to be older when worms are maturing (puppies infected via transplacental migration will have mature worms by 14 days of age, whereas kittens infected via the transmammary route will have mature worms by 28 days of age) and have therefore had more time to grow and develop their body condition before the problems associated with mature worms may be seen. For this reason the clinical signs associated with mature worms are often far less severe, and may possibly even go unnoticed in kittens when compared to puppies (Overgaauw,

1997b).

Infection with mature Toxocara is far less common in adult animals than it is in the young. According to Greve (1971) by the time a puppy has reached the age of one to two months, the probability that larvae of T. canis will develop into adult ascarids falls to a very low level, whereas the probability of somatic migration progressively increases. Particular circumstances can result in the development of patent infection in adult dogs. These circumstances include consumption of infected paratenic hosts, immunosuppression or hormonal changes (Fahrion et al ., 2008).

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Little information is available on the clinical effects of Toxascaris leonina infection in dogs and cats. However, T. leonina is probably the least pathogenic of the ascarids of dogs and cats because there is limited larval migration within the definitive host, no prenatal or transmammary infection and a long prepatent period that allows puppies and kittens to grow before the worms develop to adult size (Parsons, 1987).

Patent Toxocara infection in dogs and cats can be tentatively diagnosed from the medical history, particularly the lack of an appropriate anthelmintic schedule and the clinical symptoms. Confirmation of the diagnosis is best made by examining the faeces using a flotation technique, the specific gravity of the flotation solution should be at least 1.18 and centrifugation is preferred (Dryden, 1996).

Eggs of Toxocara are very resistant to adverse environmental conditions and can remain infective for years (Overgaauw, 1997b). Hence the best method of preventing infection with this parasite is to reduce environmental contamination. A decrease in contamination can be achieved by cleaning up faeces regularly, and by the use of strategic anthelmintic treatment of puppies, kittens, nursing bitches and queens. In puppies anthelmintic treatment should first be administered at two weeks of age, and because milk transmission occurs continuously for at least five weeks post-partum, repeated treatments are necessary. Larvae that reach the intestine need at least two weeks to mature and start passing eggs, therefore the treatment should be repeated every fourteen days (Overgaauw, 1997b; Bowman et al ., 2003). Reinfection can occur throughout the suckling period and treatment should be at least continued until the time when the last larvae arrive through the milk in the puppies’ intestine at seven weeks of age (Barriga, 1991). Preventive treatment in kittens should begin at six weeks, because unlike puppies prenatal infection does not occur and hence excretion

24 of eggs begins later. Bitches and queens should always be included in anthelmintic treatments at the same time as the puppies and kittens (Overgaauw, 1997b).

1.3.1.2.2 Toxocariasis

Toxocariasis is the clinical disease in humans caused by infection with T. canis or T. cati . Humans act as an accidental host in which Toxocara larvae generally do not develop but migrate. Humans become infected by ingesting infective Toxocara eggs from contaminated soil, from unwashed hands, consumption of raw vegetables or from the ingestion of larvae in undercooked organ and muscle tissue of infected paratenic hosts, such as chickens, cattle and sheep (Overgaauw, 1997a).

While most people infected with Toxocara do not develop overt clinical disease

(Schantz, 1989), three clinical syndromes have been associated with Toxocara infection in humans; visceral larva migrans (VLM), ocular larva migrans (OLM) and covert toxocariasis (Taylor and Holland, 2001).

Visceral Larva Migrans syndrome results from the migration of larvae and the marked, inflammatory immune response by the host. The clinical signs associated are usually non-specific and are generally the result of the host’s immune response

(Overgaauw, 1997a). Visceral larva migrans is mainly diagnosed in children between one to seven years of age (mean age two years), and if subsequent exposure to

Toxocara eggs is avoided, the disease is usually self-limiting (Overgaauw, 1997a).

Ocular Larva Migrans syndrome is usually caused by no more than a single migrating larva. This larva seems to induce granulomatous retinal lesions, which are characterised by complaints of loss of visual acuity, squinting, ‘seeing lights’ and can

25 in a minority of cases result in total blindness of one or more rarely both eyes

(Overgaauw, 1997a). Infection with Toxocara rarely results in concurrent ocular and systemic disease (Overgaauw, 1997a).

The term “covert” toxocariasis has been usde to describe a clinical syndrome in patients with raised titres to Toxocara and non-specific signs including abdominal pain, anorexia, vomiting, nausea, sleep and behavioural disturbances, cervical adenitis, wheezing, cough, limb pains and fever (Glickman et al ., 1987; Taylor et al .,

1987). Other associated illnesses such as social, learning and behavioural abnormalities, epilepsy, asthma and transient myositis have also implicated Toxocara as the cause of these wide range of clinical signs, however the interpretations of these findings have been debated (Schantz, 1989; Taylor and Holland, 2001).

1.3.1.3 Trichuris vulpis

Trichuris vulpis is a parasite of dogs and although infected animals are usually symptomless, heavy infections may be associated with bouts of diarrhoea, sometimes containing mucous and fresh blood, alternating with periods during which normal stools are passed (Bowman et al ., 2003). Trichuris vulpis has a long prepatent period

(Bowman et al ., 2003), so it usually affects older dogs. There is no age increased immunity against infection, consequently adult dogs may suffer clinical signs.

Detection of characteristic eggs, together with appropriate clinical signs, enables a diagnosis of trichuriasis to be made. However, given that the eggs are often passed intermittently a repeated faecal exam may be necessary. The eggs are also heavier than some other helminths and a flotation solution with a specific gravity of about 1.4

26

(e.g. saturated sodium nitrate) is often needed for detection (Dunsmore and Shaw,

1990).

The most important feature regarding infection with Trichuris is the longevity of the eggs in the environment; up to five years survival has been recorded in a humid and warm climate (Dunsmore and Shaw, 1990). Once Trichuris eggs have been passed, under optimal conditions they will only reach infectivity in three to four weeks

(Dunsmore and Shaw, 1990), hence removal of all faeces frequently will avoid environmental contamination and reinfection. If an area has been contaminated with eggs, it must be expected that reinfection of dogs using this area is likely to occur for some time. This is of particular importance in a refuge situation. Infections can be treated with a variety of anthelmintics, and if dogs are confined to a contaminated environment, given that the prepatent period of this parasite is 10-12 weeks, treatment every eight weeks for a year or more must be recommended (Dunsmore and Shaw,

1990).

Trichuris vulpis has been recovered infrequently from humans (Dunn et al ., 2002).

Most reported cases in humans have occurred with children and institutionalised patients. Symptoms may range from asymptomatic carriage to diarrhoea (Hall and

Sonnenberg, 1956; Kagei et al ., 1986; Mirdha et al ., 1998).

1.3.1.4 Dipylidium caninum

The definitive hosts of Dipylidium include dogs, cats and wild carnivores; humans are occasional hosts (Molina et al ., 2003). Cysticercoids develop in fleas

(Ctenocephalides spp.) and biting lice ( Trichodectes canis ), and the dog and cat become infected through the ingestion of these intermediate hosts. Adult tapeworms

27 cause little harm in dogs and cats (Bowman et al ., 2003) and prevention of infection is by keeping pets free of fleas.

Faecal flotation is not reliable for detecting the eggs of D. caninum as the eggs are not regularly released from proglottids into the faecal stream leading to false-negative results. Necropsy is the ‘Gold standard’ diagnostic procedure but is obviously not appropriate for domestic pets (Robertson et al ., 2000).

The first report of human dipylidiasis was in 1903 (Stiles, 1993), and since then less than 100 cases have been reported in the English-language literature (Molina et al .,

2003). Most infections occur in children, and one-third of these cases involve infants that are 6 months of age or younger (Reid et al ., 1992). Young children may ingest infected fleas, or the larval tapeworm may be transferred from the tongue of a pet to the mouth of a child by the act of licking (Jones, 1979). Infection in humans is usually asymptomatic, but some patients may show loss of appetite, indigestion, abdominal pain, poor weight gain or diarrhoea (Turner, 1962; Chappell et al ., 1990).

1.3.1.5 Spirometra erinacei

Species from the families Canidae and Felidae, including the dog and cat, are the definitive hosts of Spirometra erinacei . Infection of the definitive host has no clinical significance (Georgi, 1987). The first intermediate hosts are freshwater crustaceans

(copepods), while the second intermediate or paratenic hosts may be a wide range of animals (snakes, frogs, hedgehogs, poultry, pigs, echidnas, platypuses and occasionally humans) (Whittington et al ., 1992; Pampiglione et al ., 2003).

28

Humans can become infected via a number of routes: a) by drinking water containing parasitised copepods; b) by eating raw or insufficiently treated meat of an intermediate parasitised host; c) by contact with the flesh of a second intermediate host (in some countries, it is a custom to apply the flesh of animals to wounds) (Pampiglione et al .,

2003). In humans the plerocercoids are usually found subcutaneously or occasionally in various other parts of the body: in the lower limbs, the wall of the abdomen or the chest, the scrotum, breasts, eyes and brain (Pampiglione et al ., 2003).

1.3.1.6 Echinococcus granulosus

1.3.1.6.1 Infection in dogs

The sheep strain (G1) of Echinococcus granulosus is the only strain thought to exist on mainland Australia (Thompson and McManus, 2002). There are a number of known intermediate hosts which include sheep, cattle, pigs and macropods; it is of course also infective to humans. The definitive hosts in Australia include the dog, fox and dingo (Thompson and McManus, 2002). Several transmission cycles occur on mainland Australia (Eckert et al ., 2001b). One cycle involves domestic sheep as the major intermediate host. Within this cycle cattle and pigs may also act as potential accidental intermediate hosts, however it would seem that they play only a small role in transmission as the cysts are usually sterile. The other cycle involves a number of species of macropod marsupials (kangaroos and wallabies). The two cycles are known to interact through the definitive hosts (domestic dogs, wild dogs, dingoes and red foxes) (Thompson and McManus, 2002).

Up to the early 1970’s E. granulosus was common in rural dogs, particularly in eastern Australia, where rates of carriage were often over 20% (Schantz et al ., 1995).

However, subsequently, there has been an overall decline of infection in rural dogs

29

(Jenkins, 1996) which has been attributed to two main factors. Firstly the introduction of cheap commercially available dried dog food in the 1960’s, and secondly the discovery of praziquantel, a safe highly efficient cestocidal drug (Jenkins, 2005). The availability of these two products combined with public education, eventuated in a substantial decline of echinococcosis in rural dogs (Jenkins, 2005).

There is no recent information on the prevalence of infection with E. granulosus in livestock, domestic dogs and humans in Australia (Schantz et al ., 1995; Eckert et al .,

2001b; Jenkins, 2005). However, focal areas with high levels of infection in domestic dogs still remain in Australia (Jenkins et al ., 2006). Schantz et al . (1995) stated that it would be difficult to make an accurate assessment of the current distribution of this parasite in Australia as data has never been collected at a national level, and although a number of surveys have been carried out in the past, they have been of short duration and carried out irregularly.

Infection of canids with Echinococcus cannot be diagnosed by microscopic egg detection in faecal samples, because eggs are indistinguishable from those of different

Taeniid cestodes. Furthermore, proglottid/egg excretion is often irregular. Hence unfortunately the “gold standard” for diagnosis remains post mortem detection of the parasite (Eckert et al ., 2001a), but given that this is not a viable technique in many epidemiological surveys other methods have evolved.

Traditionally, the standard method used for diagnosing infection in dogs has been the use of arecoline purging. Arecoline hydrobromide causes rapid and strong contractions of the smooth muscle of the intestine, as well as paralysing the worm itself. The subsequent purgation carries the worms out with the faeces (Eckert et al .,

2001a). This drug has several major disadvantages: it can be lethal for very young or

30 old dogs, it may cause pregnant dogs to abort, dogs with small infections may not pass worms, a portion of arecoline-treated dogs may purge incompletely or not at all, the procedure is said to be very time consuming and purging results are always an under- representation of the true infection situation (Jenkins, 2005).

Currently there are several enzyme-linked immunosorbent assays (ELISA) described for the detection of coproantigens released by E. granulosus (Allan et al ., 1992;

Deplazes et al ., 1992; Sakai, 1996; Ahmad and Nizami, 1998; Jenkins et al ., 2000;

Benito and Carmena, 2005). This technique not only considerably improved both the sensitivity and specificity for detecting infected dogs (Benito and Carmena, 2005), but also had the advantage over serum antibody detection in the high probability of correlation with current infections (Eckert et al ., 2001a). Other advantages of this method include that it permits the detection of the parasite during the prepatent period,

ELISA values decrease to negative values 2 to 4 days after the elimination of the worms (Jenkins et al ., 2000) and the ELISA results correlate well with the worm burden in the intestine (Craig et al ., 1995).

A recent advance has been the development of a copro polymerase chain reaction

(PCR) for the detection of parasite derived DNA from faeces with reportedly high sensitivity and specificity (Abbasi et al ., 2003; Stefanic et al ., 2004). Lahmar et al .

(2007) made a comparison between arecoline purgation, coproantigen ELISA and coproPCR with necropsy in prepatent infections, and concluded that, surveillance of canine echinococcosis can be achieved effectively by the application of coproantigen

ELISA and specific confirmation of copropositives by coproPCR (Lahmar et al .,

2007).

31

1.3.1.6.2 Hydatid disease

Humans can act as an accidental dead-end intermediate host of E. granulosus and infection gives rise to a condition known as cystic hydatid disease. Humans become infected following the ingestion of eggs through either direct contact with definitive hosts, or indirectly through contaminated food, water and objects. Coprophagic flies and other animals may serve as mechanical vectors of the eggs (Gemmell, 1958) resulting in greater dispersal and environmental contamination. Following ingestion, oncospheres hatch from the egg, penetrate the intestinal mucosa, and enter the blood stream. While circulating in the blood they may become trapped in visceral organs, most commonly the liver and lung, and less commonly in muscle, bone, kidneys, eyes and brain. When the site is established the oncospheres develop into cysts containing multiplying larvae (Tan, 1997). Most patients are asymptomatic and the cyst is discovered accidentally on radiography (Tan, 1997); although in some instances infection may result in severe disease or even death (Pawlowski et al ., 2001).

Treatment usually necessitates surgery which is sometimes unsuccessful and needs to be repeated. Albendazole can be used as an adjunct to treatment, especially in inoperable cases or prior to surgery.

Human cases of hydatid disease continue to occur in Australia. During a four year period between January 1991 and December 1994, the National Notifiable Diseases

Surveillance System of Australia recorded 170 human cases of hydatid disease.

Notifications were received from all States and Territories, with the majority of reports from Queensland, New South Wales and Victoria (Longbottom and

Hargreaves, 1995). This, however, is probably a gross underestimate given the findings of Jenkins and Power (1996) which suggested that human hydatid infection was seriously under-reported in Australia. In their retrospective study, data were

32 collected on cases of hydatid diseases occurring between 1987 and 1992 from 38 hospitals or health services in New South Wales and four hospitals in the Australian

Capital Territory. They reported 195 new cases, compared with a total of 40 officially notified cases during the same period. Hydatid disease is no longer notifiable in

Australia (Jenkins, 2005).

Gemmell (1958) reported that one of the main factors influencing transmission of

Echinococcus is climate. Eggs of E. granulosus are sensitive to desiccation however they can remain infective for up to 1 year in a moist environment at lower temperature ranges. In regions with temperatures above 30˚C combined with rainfall of less than

25mm per month the incidence of hydatidosis is low. Much of Australia is unsuitable climatically for the maintenance of the life-cycle of E. granulosus and the parasite is hypothetically restricted to certain regions which include the south western corner, large parts of the eastern coast and Tablelands on the mainland (Kumaratilake and

Thompson, 1982).

1.3.2 Protozoa

1.3.2.1 Giardia duodenalis

1.3.2.1.1 Dog and cat infection

Reported prevalences of Giardia in dogs and cats worldwide have ranged from 2.4% to 80% (Swan and Thompson, 1986; Collins et al ., 1987; Bugg et al ., 1999; Hill et al .,

2000; McGlade et al ., 2003b; Asano et al ., 2004; Huber et al ., 2005; Papini et al .,

2005; Shukla et al ., 2006; Inpankaew et al ., 2007; Liu et al ., 2008; Meireles et al .,

2008). Although Giardia is common in dogs and cats, it is rarely associated with clinical disease. Barr and Bowman (1994) surveyed a variety of canine populations for the presence of Giardia and reported a prevalence of approximately 10% in well cared

33 for dogs, 30-50% in pups and up to 100% in dogs from breeding establishments and kennels. If clinical giardiasis manifests, it is usually associated with young animals and those in kennel or cattery situations (Robertson et al ., 2000), where the effects of overcrowding may cause stress and exacerbate the effects of an infection (Thompson,

2004). The most consistent clinical sign of giardiasis is diarrhoea, which may be acute or chronic, and self-limiting, intermittent, or continuous (Barr and Bowman, 1994).

A G. duodenalis vaccine, produced from trophozoites isolated from sheep, is available for dogs and cats in North America (Olson et al ., 2000). Puppies and kittens inoculated with the vaccine subcutaneously and subsequently challenged did not develop clinical signs of giardiasis. These authors demonstrated a reduction or elimination of intestinal trophozoites and faecal cyst excretion, while vaccinated animals had higher weight gains compared to non-vaccinated animals (Olson et al .,

1996; Olson et al ., 1997). Furthermore, the vaccine has been used as a therapeutic agent in dogs chronically ill with giardiasis which had not responded to chemotherapeutic drugs, and vaccination resulted in the cessation of clinical signs and faecal cyst shedding (Olson et al ., 2001). However, a number of other studies have failed to demonstrate a significant effect of the vaccine on infected animals (Payne et al ., 2002; Stein et al ., 2003; Anderson et al ., 2004).

Diagnosis of Giardia by traditional microscopic methods following the application of faecal concentration techniques, especially zinc sulphate flotation and centrifugation remain a reliable indicator of infection (Zajac et al ., 2002). The sensitivity of this procedure increases from approximately 70% on examination of a single sample up to

95% if the tests are conducted over a 3-5 day period (Irwin, 2002). Direct immunofluorescence microscopy has also improved the sensitivity for detecting and

34 quantifying faecal cysts and may allow for more accurate determination of prevalence and cyst excretion intensities compared to conventional microscopy (O'Handley,

2002). There are several ELISA-based methods available that detect coproantigens, and these work well but are relatively expensive. More recently, PCR-based detection of Giardia directly from faecal samples has been used (McGlade et al ., 2003b). A

PCR used in conjunction with sequencing or restriction fragment length polymorphism (RFLP) can provide information on the genotype or species of Giardia present (Thompson, 2004).

1.3.2.1.2 Human infection

Dogs and cats carry strains of Giardia which are potentially infective to humans

(Traub et al ., 2003). Giardia duodenalis is transmitted by the faecal-oral route, producing environmentally resistant cysts that are voided in the faeces and transmitted directly or via water or food, to another host. Symptoms of Giardia infection in humans may include acute or chronic diarrhoea, dehydration, abdominal pain, nausea, vomiting and weight loss. The number and severity of symptoms can be highly variable and many people remain asymptomatic (Thompson and Monis, 2004).

Understanding the zoonotic potential of dogs and cats infected with Giardia requires knowledge of the genetic diversity that occurs within G. duodenalis . Most of the assemblages of G. duodenalis seem to be host specific or have a limited host range.

To date only two of the assemblages (A and B) are known to have genotypes that occur in humans. Assemblage B genotypes seem to be human specific however there are related genotypes within Assemblage B that have been isolated from other animals including dogs (Thompson and Monis, 2004). Group AI infections consist of a mixture of closely related animals (including dogs and cats) and humans, and perhaps

35 have the greatest zoonotic potential. Genotypes from Group AII were thought to be composed of entirely human isolates until recently when Traub et al . (2004) recovered

Giardia from this subgroup from dogs in a remote tea growing community in Assam,

India.

Analysis of genetic data has demonstrated that the major lineages of G. duodenalis are as divergent from each other as they are from other species of Giardia ; and hence provides strong grounds for considering these lineages distinct species (Monis et al .,

1999; Thompson and Monis, 2004). Thompson and Monis (2004) have made a proposal for the revised taxonomy of G. duodenalis , in which they suggested dividing the assemblages into a number of species which have been included in Table 1.3.

Table 1.3 Assemblages of G. duodenalis (based on the allozymic analysis of

multilocus enzyme electrophoresis by Monis et al ., (1999); Monis et al . (2003) and

the subsequent species names proposed by Thompson and Monis (2004))

Assemblage Proposed species Host

A G. duodenalis Humans and other primates, dogs, cats,

livestock, rodents and a variety of wild

mammals

B G. enterica Humans and other primates, dogs

C G. canis Dogs, cats

D Dogs, cats

E G. bovis Hoofed livestock

F G. cati Cats

G G. simondi Rats

36

1.3.2.2 Enteric coccidians

1.3.2.2.1 Cryptosporidium

Dogs and cats are most commonly infected with what appear to be predominantly host-adapted species of Cryptosporidium : C. canis and C. felis (Morgan et al ., 1998;

Abe et al ., 2002b; Fayer et al ., 2006). The extent to which dogs and cats may act as mechanical vectors for other Cryptosporidium species is also yet to be determined, however the potential was recently demonstrated with the recovery of oocysts of both

C. baileyi and C. muris from cat faeces (McGlade et al ., 2003a). Hajdusek et al .

(2004) also recently recovered the oocysts of C. meleagridis from the faeces of a dog.

Healthy immunocompetent dogs and cats usually remain asymptomatic to infection with Cryptosporidium and the infection is generally self-limiting. In contrast, signs of disease usually manifest in animals with compromised or poorly developed immune systems. Therefore, very young, very old and otherwise immunosuppressed individuals (e.g. dogs with canine distemper and cats with feline leukaemia virus or feline immunodeficiency virus infections) (Miller et al ., 2003) or in animals with concomitant infection with other intestinal pathogens (Barr, 1997) are most severely affected. Clinical signs of cryptosporidiosis generally include chronic anorexia, weight loss and persistent diarrhoea, usually of a small intestinal nature (Barr, 1997).

Oocysts of Cryptosporidium spp. are immediately infectious when passed by the host.

Because these parasites are transmitted in nature primarily by the faecal-oral route, it could be expected that higher prevalences will be seen associated with crowded and unsanitary environmental conditions (Lappin, 2005) which may be the case in many refuges, breeding establishments and pet shops.

37

As a consequence of their small size and the often limited number in faeces of infected animals, oocysts of Cryptosporidium spp. are easily missed under light microscopy, even when concentrated via a flotation technique. Elliot et al . (1999) described a malachite green negative staining technique which was shown to improve the detection of oocysts in faeces by microscopic examination. Although microscopy provides the advantage of direct visual confirmation of the presence of

Cryptosporidium oocysts, it does not allow for the differentiation of morphologically similar species. In contrast PCR is highly sensitive and the amplified DNA can be studied further to determine differences in strains or species using genetic techniques.

Determining the species of Cryptosporidium involved in an infection is imperative to understanding the epidemiology and zoonotic potential of this parasite.

Cryptosporidium canis and C. felis have been implicated as causative agents of diarrhoea in humans with AIDS. However, the pathogenicity in immunocompetent patients is currently unclear because all reported infections have been in asymptomatic individuals (Pedraza-Diaz et al ., 2001; Xiao et al ., 2001; Hajdusek et al ., 2004).

Human infection associated with contact with infected dogs and cats has been reported but is thought to be unusual (Lappin, 2005). In one study, cat ownership was not statistically associated with cryptosporidiosis in HIV infected people (Glaser et al .,

1994). In contrast to this, Morgan et al . (2000) found that three of six immunodeficient patients infected with C. felis had reported having a cat as a pet and it was concluded that these patients may have acquired their infection from their pets.

In addition to this, Morgan et al . (2000) proposed that for the remaining three patients where no pets were recorded it was possible that these patients had been exposed to cats at some time prior to the onset of clinically apparent cryptosporidiosis.

38

1.3.2.2.2 Isospora

With the frequent use of anthelmintics in pet dogs and cats , Isospora spp. are currently some of the most commonly diagnosed parasites on faecal examination. Generally, infected dogs and cats remain asymptomatic, however if clinical signs manifest it is usually in puppies and kittens, and diarrhoea is the most common presentation

(Lindsay et al ., 1997a). It should be noted that, even if oocysts are found in association with diarrhoea, they cannot be conclusively deemed to be the cause of the diarrhoea unless other potential causes have been vigorously excluded (Kirkpatrick and Dubey, 1987).

Four species of Isospora are known to infect dogs; I. canis , I. ohioensis , I. neorivolta and I. burrowsi . Oocysts of I. canis can be differentiated from the larger oocysts of the other Isospora species, however the oocysts of I. ohioensis , I. neorivolta and I. burrowsi are indistinguishable from each other and are collectively referred to as the

Isospora ohioensis-complex (Conboy, 1998). Generally I. canis and I. ohioensis are the species most frequently associated with diarrhoea (Lindsay et al ., 1997a). It has been reported that stray dogs are more likely to be infected than are dogs with owners because stray dogs must hunt for food and therefore have more exposure to infected paratenic hosts (Lindsay et al ., 1997a).

There are several reports which have implicated I. ohioensis as a pathogen causing enteric disease in young dogs (Dubey, 1978; Olson, 1985; Conboy, 1998). Puppies under four months of age are said to be at greatest risk, especially in large kennel situations, such as dog breeder facilities or refuges (Conboy, 1998). Clinical signs consist of diarrhoea, which may be bloody, with varying degrees of abdominal pain,

39 anorexia, anaemia, and weight loss. Respiratory and neurological signs have also been reported, and in rare cases death has eventuated (Dubey, 1978; Olson, 1985; Conboy,

1998). Yet experimental evidence in support of the role of I. ohioensis as a pathogen in dogs has been inconclusive (Conboy, 1998). Diarrhoea was induced in neonatal puppies exposed to oocysts, but no clinical disease was observed in similarly exposed weaned puppies or young dogs (Dubey, 1978). Conboy (1998) speculated that clinical disease mainly occurs in immunologically naïve young dogs exposed to large doses of

I. ohioensis .

There are two species of Isospora known to infect cats: I. felis and I. rivolta .

Experimentally I. rivolta can cause diarrhoea in newborn kittens, yet no disease was seen in weaned kittens inoculated with up to 10 5 oocysts (Lindsay et al ., 1997a).

Experimental studies indicate that I. felis is moderately pathogenic for six to 13 week old kittens given 1 to 1.5 x 10 5 oocysts. Soft, mucoid faeces were observed in kittens eight days after infection. In contrast to older kittens, four week old kittens may develop severe disease characterised by signs of enteritis, emaciation, and even death when given 1 x 10 5 oocysts (Bowman et al ., 2002).

Species of Isospora are readily diagnosed on routine faecal examination using any of the faecal flotation methods and most small animals are believed capable of spontaneously eliminating infections (Lindsay et al ., 1997a).

1.3.2.2.3 Sarcocystis spp.

Sarcocystis has an obligatory two-host life cycle; an intermediate host (herbivorous animals) and the final host (carnivores) (Dubey, 1976). Species-specific prey-predator life cycles have been demonstrated for cattle-dog ( S. cruzi ), cattle-cat ( S. hirsuta ),

40 sheep-dog ( S. tenella, S. arieticanis ), sheep-cat ( S. gigantean, S. medusiformis ), goat- dog ( S. capracanis ) as well as others (Dubey, 1976). Herbivores become infected after ingesting sporocysts or oocysts in the faeces of the definitive hosts, which in turn acquire infection by ingesting sarcocysts in the tissues of intermediate hosts (Dubey,

1976). The domestic dog/cat becomes infected when it ingests uncooked meat and infection is of little clinical significance (Bowman et al ., 2003). On the contrary,

Sarcocystis infection in the intermediate host can cause severe pathology and is of economic importance, for example infection can cause abortion, acute fatal illness or poor growth in cattle, sheep and goats (Dubey, 1976; Frelier, 1977; Dubey and Fayer,

1983; Dubey et al ., 1986; Dubey, 1988; Fayer and Dubey, 1988).

1.3.2.2.4 Hammondia

1.3.2.2.4.1 Hammondia heydorni

The dog is the definitive host of Hammondia heydorni and cattle usually act as the intermediate host (Dubey et al ., 2002), although other animals (such as sheep, goat, buffalo, deer, and moose) can also act as intermediate hosts (Dubey et al ., 2002;

Dubey et al ., 2004). The unsporulated oocysts of H. heydorni and Neospora caninum , and the feline coccidians, Toxoplasma gondii and Hammondia hammondi , are all morphologically identical (Reichel et al ., 2007). Dogs can mechanically transmit the oocysts of T. gondii and H. hammondi (Lindsay et al ., 1997b; Schares et al ., 2005).

Molecular techniques, based on PCR, can be used to differentiate the species of coccidia present in dog’s faeces (Reichel et al ., 2007). Dogs are at risk of infection with H. heydorni when fed raw meat (Dubey et al ., 2003; Schares et al ., 2005) and studies have reported that infection can result in diarrhoea in normal and immunosuppressed dogs (Blagburn et al ., 1988; Schares et al ., 2005; Abel et al .,

2006).

41

1.3.2.2.4.2 Hammondia hammondi

Hammondia hammondi has a heteroxenous life cycle involving the cat as the definitive host and rodents as the intermediate host (Frenkel and Dubey, 1975). This coccidian has not been associated with any disease manifestation in any of the hosts, but is epidemiologically significant due to its close resemblance to T. gondii

(Sreekumar et al ., 2005).

1.3.2.2.5 Toxoplasma gondii

Toxoplasma gondii is a ubiquitous coccidian with a worldwide distribution (Dubey and Lappin, 1998). Cats are the only known definitive host of T. gondii in which completion of the enteroepithelial cycle (sexual phase) takes place and results in the passage of environmentally resistant unsporulated oocysts in the faeces (Lappin,

2005). Generally, only about one per cent of cats in a population are found to be shedding oocysts at any given time. Oocysts are shed for only a short period (1-2 weeks) in the life of the cat; however, the enormous numbers shed result in widespread contamination of the environment (Hill and Dubey, 2002). Oocyst sporulation occurs within one to five days depending on environmental conditions

(Bowman et al ., 2002), and sporulated oocysts are infectious to most warm-blooded vertebrates (Lappin, 2005). After infection, an extraintestinal phase develops, which ultimately leads to the formation of tissue cysts containing the organism. Infection by

T. gondii occurs after ingesting sporulated oocysts, after ingesting tissue cysts, or transplacentally (Lappin, 2005).

Infection rates in cats are said to reflect the rate of infection in the local population of animals they prey upon (Hill and Dubey, 2002). However, a confounding factor in this

42 assumption is the practice of some cat owners feeding their pet’s raw or undercooked meat. Although most infected cats remain asymptomatic, some may develop fever and clinical signs of dyspnoea, polypnoea, icterus and signs of abdominal discomfort

(Bowman et al ., 2002). Disease in congenitally infected kittens can be severe and fatal. The most common clinical signs are anorexia, lethargy, hypothermia and sudden death (Bowman et al ., 2002).

1.3.2.2.5.1 Human infection

Humans become infected by ingesting tissue cysts in undercooked or raw meat or by ingesting food or water contaminated with oocysts from infected cat faeces (Bowman et al ., 2002). The role of cat ownership and human exposure to T. gondii is not completely clear at present. Of the studies conducted which explore the relationship between cat ownership or cat exposure and the prevalence of infection in humans, some have found no positive association, while others have (Bowman et al ., 2002). It appears that human infection is most likely to result from the consumption of raw or poorly cooked meat rather than from ingesting oocysts from a cat (Angulo et al .,

1994).

It has been estimated that nearly one-third of humans have been exposed to T. gondii

(Hill and Dubey, 2002), yet most infected immunocompetent humans remain asymptomatic; however occasionally self-limiting fever, lymphadenopathy, and malaise may occur (Lappin, 2005). Transplacental infections may give rise to a wide spectrum of clinical signs that include stillbirth, hydrocephalus, hepatosplenomegaly, and retinochoroiditis. Chronic tissue infection in people can be reactivated by immunosuppression, leading to disseminated and severe clinical illness (Lappin,

2005). Toxoplasmosis is said to rank high on the list of diseases which lead to death in

43 patients with acquired immunodeficiency syndrome (AIDS); and according to one paper published in 1992 approximately 10% of patients with AIDS in the USA and up to 30% in Europe at the time of investigation were said to have died from toxoplasmosis (Luft and Remington, 1992).

Walpole et al . (1991) conducted a prospective serological study of randomly selected pregnant women and their newborn infants in Perth, Western Australia. Of the 10,207 women screened at their first antenatal visit 35% had antibodies for T. gondii . The rate of maternal infection in susceptible pregnancies was determined to be 1.6 per 1000, and the maternal-foetal transmission rate was estimated at no greater than 24%. More recently Karunajeewa et al . (2001) determined the seroprevalence of a group of women at an antenatal clinic in a Melbourne obstetric hospital. Of the 308 women screened 23% demonstrated evidence of previous exposure to T. gondii . Both of these surveys suggest that the prevalence of T. gondii exposure in humans in Australia is similar to that in the USA and the UK (16-40%), whereas in Central and South

America and continental Europe, estimates of infection range from 50 to 80% (Dubey and Beattie, 1988). Unfortunately, risk factor analysis was not carried out in either of the above Australian surveys.

1.3.2.2.6 Neospora caninum

Neospora caninum is a major pathogen of cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep and deer (Dubey, 2003). Dogs (McAllister et al ., 1998) and also coyotes (Gondim et al ., 2004) have been described as definitive hosts, excreting oocysts, but dogs (and perhaps other carnivores) can also be intermediate hosts (Wanha et al ., 2005). Dogs become infected after consuming raw meat (McAllister et al ., 1998; Lindsay et al ., 1999).

44

Neospora undergoes a life cycle involving three principal stages. Firstly, oocysts are shed in the faeces of dogs, following ingestion of bradyzoites by the dog. Secondly, bradyzoites, which multiply slowly, are found in tissue cysts in the central nervous system, both in the canine definitive host and in a wide range of intermediate hosts.

Thirdly, tachyzoites, the rapidly multiplying stage, trigger lesion development by multiplying in and rupturing cells in the definitive host and intermediate hosts (Buxton et al ., 2002).

The most severe cases of neosporosis occur in young, congenitally infected puppies

(Dubey, 2003). Dogs under six months of age are often afflicted by an ascending paresis or paralysis of the hind-limbs. The disease may be localised or generalised, with generalised infections being more common in older dogs. In disseminated infections clinical signs may include difficulty in swallowing, myocarditis, dermatitis and pneumonia (Reichel, 2000).

Neospora infection was retrospectively diagnosed in Australia in canine tissue from

1971 (Munday et al ., 1990), however the first case of clinical neosporosis was only diagnosed in 1993 (Gasser et al ., 1993). Barber et al . (1997) surveyed 421 dogs from across Australia using an indirect fluorescent antibody test and found that 9% were seropositive for Neospora (Melbourne, 5%; Sydney, 12%; Perth, 14%).

Wouda et al . (1999) compared data from farm dogs with those examined at a university clinic, which originated mainly from urban areas. They found a significantly higher proportion of seropositive animals in the rural population (23.6% compared to 5.5%). Additionally, the seropositivity was strongly correlated with a

45 high prevalence of antibodies to N. caninum in cattle on their respective farms. On farms without dogs, the seroprevalence in cattle was significantly lower than on farms where dogs were present. Several other authors have described similar observations

(Sawada et al ., 1998; Basso et al ., 2001; Antony and Williamson, 2003).

Cattle become infected after the ingestion of oocysts; adult cattle generally show no signs of clinical disease but the parasite can be transmitted vertically, resulting in abortions or congenitally infected progeny (Dubey, 2003). The parasite can persist over several generations in cattle, thus serving as a reservoir for Neospora -infections of dogs. The economic impact of N. caninum in cattle in Australia has been estimated at AUD $85 million per annum for the dairy and AUD $25 million for the beef cattle industry (Ellis, 1997).

1.4 Risk factors for parasitism

Identification of the risk factors associated with parasitism is vital in understanding the epidemiology of parasites. Risk factor identification gives insight into the dynamics of transmission and potential reservoirs; it allows for the implementation of targeted and cost effective control programs, particularly with respect to education and strategic use of anthelmintics. Risk factors (see Table 1.4) noted in the past have been known to change as the habits of humans and their pets alter, this may in turn lead to the emergence or re-emergence of parasitic disease or new pathways for transmission

(Robertson et al ., 2000). A major risk factor noted in the past, particularly with regard to nematode and protozoan infection, has been age. Higher parasite prevalences are often noted in younger animals most likely because of their immature immune systems, the unique routes of infection of some parasites and behavioural differences.

Factors associated with over-crowded conditions such as breeding kennels, refuges,

46 and multi-animal households have also been associated with a higher prevalence of nematode and protozoan parasites which may be a reflection of poor levels of care, unhygienic surroundings and increased contact with infected animals, especially puppies, kittens and stray dogs. Entire males have also been shown to harbour a higher prevalence of some parasites. This may be related to sex associated hormones which directly influence the immune system and may render entire males more susceptible to infection or gender related behavioural differences, especially with regard to the roaming patterns of male dogs (Maarschalkerweerd et al ., 1997; Neilson et al ., 1997).

Table 1.4 Risk factors associated with the presence of gastrointestinal parasites in

dogs and cats

Parasite Risk Factors of significance Reference

Hookworm Animals aged less than 6 months (Oliveira-Sequeira et al ., 2002)

Entire males versus females (Pandey et al ., 1987)

Animals kept at refuges (Gorski et al ., 1996; Bugg et al .,

1999; McGlade et al ., 2003a)

Stray versus pets (Prociv et al ., 1994)

Summer and Autumn (Oliveira-Sequeira et al ., 2002)

Animals allowed to hunt (paratenic hosts) (Bowman et al ., 2003)

Toxocara Animals aged less than 3-6 months (Else et al ., 1977; Rojekittikhun

et al ., 1998; Luty, 2001;

Habluetzel et al ., 2003)

(Tassi and Widenhorn, 1977;

Animals aged less than 2 years Visco et al ., 1977; Kirkpatrick,

1988; Oliveira-Sequeira et al .,

2002; Rubel et al ., 2003)

Adult male dogs (Luty, 2001; Ramirez-Barrios et

al ., 2004; Rubel et al ., 2003)

47

Entire cats Overgaauw, 1997b)

Rural dogs as opposed to urban companion dogs (Habluetzel et al ., 2003)

Increased number of dogs/ breeding kennels (Gothe and Reichler, 1990)

Animals allowed to hunt (paratenic hosts) (Overgaauw, 1997b)

Trichuris Dogs aged greater than 6 months (Visco et al ., 1977; Overgaauw

and Boersema, 1996)

Kennel dogs versus pet dogs (Vanparijs et al ., 1991)

Dipylidium Animals with flea/louse infestations (Bowman et al ., 2003) caninum

Spirometra Adult animals (Wilson-Hanson and Prescott,

1982)

Animals allowed to hunt and scavenge (Meloni et al ., 1993; Wilson-

(paratenic hosts) Hanson and Prescott, 1982)

Animals in the vicinity of a water source (Shaw et al ., 1983)

(river/pond)

Echinococcus Adult male dogs (Mehrabani et al ., 1999; Parada

et al ., 1995)

Lack of treatment with praziquantel (Parada et al ., 1995)

Rural stray as opposed to urban stray dogs (Pandey et al ., 1987; Pandey et

al ., 1988; Parada et al ., 1995)

Dogs fed offal (Jenkins et al ., 2006)

Dogs used for hunting (Thompson et al ., 1988)

Climate (Gemmell, 1958)

Giardia Animals aged less than 6 months (Bugg et al ., 1999; Itoh et al .,

2001)

Animals aged less than 2 years (Swan and Thompson, 1986;

Kirkpatrick, 1988; Arashima et

al ., 1992; Savini et al ., 1993)

Original place of purchase: pet shops or kennels (Itoh et al ., 2001)

48

Animals kept at breeding establishments or (Barr and Bowman, 1994; Bugg

refuges et al ., 1999; McGlade et al .,

2003a; Swan and Thompson,

1986)

Multi-animal households {Bugg, et al .,1999; McGlade et

al ., 2003a)

Cryptosporidium Crowded and unsanitary environmental (Lappin, 2005)

conditions

Isospora Animals less than one year of age (Barutzki and Schaper, 2003)

Overcrowded conditions (e.g. refuges and (Conboy, 1998)

breeding facilities)

Animals allowed to hunt (paratenic hosts) (Lindsay et al ., 1997a)

Sarcocystis Dogs aged less than 2 years (Kirkpatrick, 1988)

Animals kept at refuges or kennels (Bugg et al ., 1999)

Animals fed raw meat (Savini et al ., 1993)

Hammondia Dogs fed raw meat (Dubey et al ., 2003; Schares et

al ., 2005)

Animals allowed to hunt (Frenkel and Dubey, 1975)

Toxoplasma Cats fed raw meat (Hill and Dubey, 2002)

Cats allowed to hunt (Meloni et al ., 1993)

Neospora Dogs from cattle farms (Wouda et al ., 1999)

Dogs fed raw meat (McAllister et al ., 1998;

Lindsay et al ., 1999)

1.5 The importance of education in parasite control and the prevention of zoonotic disease

To date no parasites of veterinary importance have been eradicated, and since infections of pets and humans are often the result of human activity, education must play a key role in their control (Robertson et al ., 2000). The value of education in reducing the incidence of hydatid disease by changing management practices (such as

49 not feeding the offal of home-killed sheep to dogs) has been well illustrated

(Thompson, 1999a; Robertson et al ., 2000). Numerous authors have emphasised the importance of education in preventing human infection with parasitic zoonoses

(Schantz, 1994; Overgaauw, 1997b; Bugg et al ., 1999; McGlade et al ., 2003a;

Robertson et al ., 2000) and veterinarians have been identified as a potential provider of this education.

At present, from the limited number of studies which have addressed education about parasites, it is evident that there is still a great need for improvement in this area. Bugg et al . (1999) reported that in Perth, many dog owners (62.5%) were aware that canine parasites could be transmitted to humans, however, only one third (34%) of these owners could provide correct information on the mode of transmission. The awareness of the zoonotic potential of some parasites was also variable; for example 70% of pet owners were aware of the zoonotic potential of Toxocara in contrast to only 1.5% for

Cryptosporidium .

Interestingly, Bugg et al . (1999) found that of the dog owners who were aware that dogs could transmit parasites to humans and could correctly identify the modes of transmission, information was obtained from veterinarians, the health department or from schools, emphasising the importance of these information sources. In contrast, incorrect information was being commonly acquired from the mass media. The fact that children, pregnant women, the elderly and the immunocompromised constitute those members of society at greatest risk of suffering the consequences of pet- transmitted zoonotic infections is often the target for emotive publicity. This can cause undue stress amongst pet owners. Inaccurate, or misrepresented data, may also be

50 used incorrectly to sell antiparasitic drugs, resulting in their overuse and thus promoting the development of resistance.

A number of authors have attempted to investigate and address areas of weakness in pet parasite education. Kornblatt and Schantz (1980) and Harvey et al . (1991) identified that although veterinarians were an effective source of information many were providing incorrect, minimal or out of date information.

It is vital to have current information on the prevalence of parasites in pets, the risk factors associated with parasitism and the degree of pet owner education. Only then can a successful control program be implemented which targets areas of identified weakness. Surveillance and education and the manipulation of risk factors through education are recognised as some of the most cost effective and simplest methods of controlling parasites in both humans and animals.

51

1. 6 The general aims of this project were to:

1. Determine the prevalence and risk factors associated with GI parasites in dogs

and cats in Australia.

2. Identify and determine the zoonotic significance of certain parasites through

the application of molecular tools.

3. Review the deworming protocols recommended by veterinarians in relation to

standard guidelines.

4. Evaluate the veterinarian’s perception of GI parasites in comparison to the

prevalence determined.

5. Determine the degree of education veterinarians are providing pet owners on

zoonoses.

6. Evaluate pet owner awareness of zoonoses.

7. Formulate recommendations based on findings relating to GI parasite

prevalence, veterinarian deworming protocols and the education of pet owners

about zoonoses.

52

Chapter 2

General Materials and Methods

53

2.1 Study Area

Australia has amongst the highest levels of pet ownership in the world with 53% of households in Australia owning a dog and/or cat. In 2005 there were approximately

3.75 million dogs and 2.43 million cats (Hill, 2006) and their distribution overlaps that of the human population (Table 2.1).

Table 2.1 2005 Statistics of Dogs and Cats in Australia (‘000) (Hill, 2006)

Species Total NSW/ ACT VIC QLD WA SA/ NT TAS

Dogs 3754 1219 894 819 338 373 111

Cats 2426 804 599 443 261 227 92

The majority of dogs and cats in Australia are kept as pets. Dogs are also kept for hunting, racing and working purposes. Animals can be sourced by the public from pet shops, breeders, refuges/pounds and ‘backyard’ litters.

The level of pet care in Australia is high and strict legislations are in place in an effort to enforce responsible pet ownership (Donelan, 2001). Compulsory dog registration and affiliated fees exist in all States and Territories (Chaseling, 2001). The importance of pets can be seen by their incorporation into community infrastructure with designated areas for exercising pets and provisions for disposing of pet excrement. In large Australians are responsible pet owners; it has been calculated that of the owned dogs and cats in Australia less than 5% cause problems requiring the attention of councils or welfare groups (Chaseling, 2001). A high level of social responsibility seems to exist with pet ownership: leash laws are abided by, it is frowned upon if one does not pick up their pet’s excrement, sterilisation is common place and so too is vaccination (Chaseling, 2001; Donelan, 2001). Although there is an element of pet

54 abandonment, most of these animals are either relinquished to a pound/refuge or if dogs are found wandering the streets they are quickly dealt with by the local municipality. As a result there are few populations of “street dogs”.

Abandoned cats have unfortunately resulted in a large population of feral cats which tend to reside on the outskirts of urban dwellings and even in remote regions of bush where they have wreaked havoc on many wildlife species. Public perception towards cat ownership has changed in recent times and the cat population is said to be declining (Chaseling, 2001; Baldock et al ., 2003). Market research has indicated that the major reason for people not owning a cat is that they ‘dislike cats’, some of this dislike may be due to the perception that cats are a threat to wildlife (Chaseling,

2001). Another reason for the decline in the cat population is the very high desexing rates amongst cats (Chaseling, 2001). There is also a growing trend of keeping cats only indoors, or if they are allowed outdoors, many pet owners use a night curfew in an effort to curb hunting practises.

2.2 Study Design

2.2.1 Overcoming the logistical problems of conducting a national study

Faced with the shear magnitude and logistics associated with conducting a national study a unique opportunity arose. The pharmaceutical company Bayer has personnel whom visit the majority of veterinary clinics in every State and Territory in Australia, in both urban and rural regions. These personnel also visit and provide pro-bono products to many animal refuges. Collaboration with Bayer meant access to all of these locations, and in return, Bayer would have access to the final results, present a positive image to the public for their active involvement in this research, and

55 contribute to the education of pet owners about parasites and their potential impact on humans.

Veterinary clinics in Australia are regularly visited by various pharmaceutical company representatives; even those clinics in the most remote regions are accessed.

The bias associated with sampling from those veterinary clinics visited by Bayer personnel was not considered substantial given that most veterinary clinics in

Australia are accessed by not only Bayer representatives, but also representatives of other pharmaceutical companies.

2.2.2 Sample design

2.2.2.1 Stratification

Australia features a wide range of climatic zones, from the tropical regions of the north, through the arid expanses of the interior, to the temperate regions of the south.

Climate and humidity can significantly influence the prevalence of certain parasites as the external environment can affect the survival and development of the parasite

(Dunsmore and Shaw, 1990). It was thus important to stratify this study to include coverage of the various climatic zones of Australia. The locations of the places sampled were categorised into one of three different climatic zones (tropical, arid or temperate).

In consideration of the collaboration with Bayer, and given the monetary and time constraints, it was decided to only sample from those locations which Bayer personnel visit, namely veterinary clinics and refuges. A list of all of the locations serviced by

Bayer was requested, and this list was then stratified according to climatic zone, urban versus rural location and veterinary clinic versus refuge. Within these various strata

56 locations where chosen at random using the random number generator option in Excel

(Excel 2002, Microsoft). Selected locations were then propositioned by a Bayer representative and if they declined involvement they were replaced by another randomly selected location. The most common reason for declining involvement was the inablitity to spare the time needed for participation.

2.2.2.2 Sample size

The number of locations sampled was determined by the Bayer personnel, with a maximum of fifteen dogs and fifteen cats being sampled from each location. The ideal sample size for estimating prevalence was calculated according to Graat et al . (1997).

Since the prevalence of a wide range of parasites in both dogs and cats were being investigated, the maximum sample size required to determine the prevalence of any given parasite within 5% with a 95% level of confidence was calculated (an expected prevalence of 50% was assumed which results in the most animals requiring to be sampled). This resulted in a target sample size of 384 for an infinite population. It was decided to investigate prevalence according to climatic zone as this was thought to have a strong influence on parasite prevalence and as the population of animals in each climatic zone is unknown (some states and territories span over more than one climatic zone) an infinite population size was used in the calculation of the sample size. However, collecting 384 dog and 384 cat faecal samples from each climatic zone was considered ambitious given the person-power and time constraints of the project.

In total, 1400 canine and 1063 feline faecal samples were collected (Table 2.2) from

59 veterinary clinics and 26 refuges distributed throughout Australia (Figure 2.1). Of the canine samples collected, 921 originated from a region with a temperate climate,

387 originated from a region with a humid climate, and 101 originated from an area

57 with an arid climate. Of the feline samples, 754 came from a temperate climate, 242 came from a humid climate, and 67 came from an arid climate.

Table 2.2 Location of the samples collected

Veterinary Refuge Total

clinic

Dog 810 590 1400

Cat 572 491 1063

Figure 2.1 Location of samples collected in related to the population distribution in Australia

58

2.2.3 Education campaign

A brochure (Appendix 1) providing information relating to the parasites which may infect dogs and cats and be potential zoonoses was given to pet owners on completion and submission of their questionnaire. It was important that the brochure was only given after the submission of the questionnaire to prevent owners using the information provided in the brochure to answer the questionnaire.

2.2.4 The Use of Questionnaire Surveys

Written questionnaires were used to collect the relevant data needed in this study. The advantage of following a written format is that it is relatively inexpensive and has the ability to be applied over a large geographical area. It also affords the respondent the convenience of completing the questionnaire in their own time. Other questionnaire formats such as telephone surveys and personnel interviews were rejected as they were more likely to lead to incomplete and less accurate data or were more time consuming when compared to written surveys (Martin et al ., 1987).

2.2.4.1 Questionnaire design and implementation

The design of the questionnaire can influence the response rate achieved by a survey, the quality of responses gained, and the reliability of conclusions drawn from a survey results (McLennan, 1999). The questionnaire format should be kept simple in order to enable the interpretation of all responses as well as maximising participant cooperation. Optimal questionnaire design can be achieved by maintaining a logical order in the sequencing of questions, making questions simple, avoiding leading questions and providing for all possible responses (McLennan, 1999).

59

In 1996 a survey was conducted to determine the prevalence of GI parasites in dogs residing in Perth (Bugg, 1996). A questionnaire targeting pet owners was composed for this study and aimed at collecting information on pet management practices and owner awareness of zoonoses. In 2001 a similar study was implemented to determine the prevalence of GI parasites in cats in Perth (McGlade, 2001). The dog and cat pet owner questionnaires used in the study of McGlade (2001) were a modified version of that composed by Bugg et al . (1996) and not only acquired information relating to GI parasites but also external parasites. The dog and cat questionnaires used in my study

(Appendices 2 and 3) only focused on collecting information on GI parasites, and were essentially based on the questionnaire of Bugg et al . (1996).

The questionnaires administered to pet owners were used to obtain information on demographic data such as breed, age, gender and management data such as frequency of contact with other dogs and cats, frequency and method of anthelmintic use, frequency of exposure to faecal material, consumption of raw and uncooked meat and predation of other animals by the pet. Information was also collected pertaining to the pet owner’s demographics, awareness of parasites and access to anthelmintic information. The information obtained from the questionnaires was necessary in order to establish the potential risk factors for parasitism in dogs and cats, as well as to identify the risk factors to humans associated with the parasitism of pet animals.

The questionnaire for veterinarians (Appendix 4) was designed for the purpose of this study and was aimed at collecting information about the perceived risk of parasitic infection in the animals visiting the clinic, the anthelmintic treatment and regimes recommended to the client for the control of GI parasites and the information they provided to their clients about these parasites. Collection of such data allowed for a

60 comparison between pet owner knowledge of dog and cat parasites and the level of education veterinarians were providing on such matters.

A data sheet (Appendix 5) was also given to each refuge so that the following information could be collected for every animal sampled: age, breed, gender, neutering status, whether or not the animal had received anthelmintics while at the refuge, if anthelmintics had been administered how long ago and the period of time which the dog/cat had been at the refuge.

2.2.4.2 Pretrial of the Questionnaires

The pet owner questionnaires and veterinarian questionnaire were initially trialed at three selected veterinary practices in south-east Queensland. This was carried out to identify any possible problems with the questionnaire and to estimate the average time taken by the respondents to complete the questionnaire. It took approximately eight minutes for all questions to be completed by the respondents. There did not appear to be any problems with the respondent’s interpretations of the questions and the questionnaires were not altered from the initial format.

2.2.5 Study Implementation

Veterinarians were instructed not to collect samples from animals that were suffering from disease or had any GI symptoms. It was suggested that samples be collected from those animals presented for routine procedures such as vaccination or sterilization. The free examination of the faecal sample, and return of the results, was emphasised as an incentive for participation in the study. On completion of the questionnaire each participant was given an information brochure (Appendix 1).

61

Samples were collected over a two year period, during three collections, each of which consisted of a three month collection period. The first collection was from

March-May 2004, the second collection November 2004-January 2005 and the final collection July-September 2005. Initially it was hoped that the timing of the sampling would allow for the investigation of any seasonal fluctuation in the prevalence of parasitism.

2.2.6 Preservation and transportation of faecal samples

Due to the prolonged time lapse between collection of the faeces and processing, it was necessary to preserve the samples. Samples were preserved in two different solutions: 10% formalin for the microscopic analysis and 20% dimethyl sulphoxide

(DMSO) in saturated salt solution for future molecular based screening. Once at

Murdoch University preserved faeces were immediately refrigerated at 4º C. The decision to use 20% DMSO for preservation over ethanol or potassium dichromate solution was made in consideration of the transporting constraints with regard to ethanol being a fire hazard and the health risks associated with handling potassium dichromate.

2.3 Parasitological techniques

The methods most commonly used to recover parasitic eggs and oocysts from faeces are flotation techniques that rely on the differences in the specific gravity (SG) of the egg(s), faecal debris, and flotation solution (Dryden et al ., 2005). The specific gravity of most parasite eggs is between 1.05 and 1.23 (David and Lindquist, 1982), and for eggs to float, the SG of the flotation solution must be greater than that of the eggs

(Dryden et al ., 2005).

62

Flotation techniques were elected over the formalin-ethyl acetate technique (Truant et al ., 1981) given the differences in sensitivity of both procedures are negligible and flotation has been shown to yield a cleaner preparation with less background and faecal debris (Bartlett et al ., 1978). Formalin fixation clears the internal structures of protozoan cysts and is said to prevent distortion commonly associated with salt solutions of high specific gravity (Bartlett et al ., 1978).

The length of formalin fixation of faecal samples has been shown to alter the effectiveness and sensitivity of flotation techniques at recovering parasite stages

(Bartlett et al ., 1978). Prolonged formalin fixation can affect the buoyancy of eggs and cysts (Elliot, personal communication). Various flotation concentrating techniques have been trialed and assessed on formalin preserved faeces at the Parasitology

Diagnostic Laboratory in the School of Veterinary and Biomedical Sciences, Murdoch

University. A modified version of the saturated salt and D-glucose solution (specific gravity 1.34) used by Henriksen and Christensen (1992) was found to be the most effective method of recovering helminth eggs and larvae (including Trichuris and

Taenia eggs) and protozoan cysts such as Giardia (Elliot, personal communication).

Each dog and cat faecal sample was initially screened using a standard sedimentation in water technique, a Malachite Green stain for Cryptosporidium oocyts (Elliot et al .,

1999) followed by saturated salt and D-glucose centrifugal flotation and microscopy.

63

Chapter 3

National study of the gastrointestinal parasites of dogs

and cats in Australia

This chapter is a published paper: PALMER, C. S., THOMPSON, R. C., TRAUB, R. J.,

REES, R. & ROBERTSON, I. D. (2008) National study of the gastrointestinal parasites of dogs and cats in Australia. Veterinary Parasitology, 151 , 181-90.

64

3.1 Introduction

The current prevalence of gastrointestinal parasites in Australian pet dogs and cats is unknown and has never been investigated on a national scale. Australia has amongst the highest levels of pet ownership in the world, with 53% of all households owning a dog or cat (Hill, 2006). The level of pet care is high and strict legislations are in place in an effort to ensure responsible pet ownership (Donelan, 2001). The pet care industry is one of the largest in Australia and contributes around four and a half billion dollars to the economy annually (Hill, 2006). Parasite prevention represents a major source of income for veterinarians, and, as per product label directions, veterinarians routinely recommend prophylactic administration of anthelmintics, at set intervals, for the control of gastrointestinal parasites. Current recommendations are based on previous studies conducted some 20-30 years ago (Tables 3.1 & 3.2), as evident by the brochures which often accompany products. These earlier studies, which reported high levels of helminth infection, generally involved small numbers of animals in a particular location and many were restricted to high risk groups (Tables 3.1 & 3.2). Of the few studies conducted in more recent times, the prevalence of helminth infection reported was significantly lower than that reported in earlier studies (Sargent, 1997;

Bugg et al ., 1999; McGlade et al ., 2003a). These recent studies all involved microscopic screening of faecal samples collected once off and as a result are likely to have underestimated the prevalence of helminths, given the intermittent nature of egg/cyst shedding, compared to studies where animals are examined postmortem or after undergoing purging (such techniques are not practical for large scale surveys).

Nevertheless, in the context of widespread prophylactic anthelmintic administration, a reduction in the prevalence of helminths in recent times is likely.

65

Current information on the regional prevalence of intestinal parasites is vital. This information is essential to local veterinarians for the development of strategies for treatment and control of parasites, and for public health authorities concerned with monitoring the zoonotic potential of infections in pets (Schantz, 1999). The purpose of this study was to determine the prevalence of gastrointestinal parasites and also identify risk factors for parasitism.

66

Table 3.1 Results of previous intestinal parasite studies in domestic dogs in Australia (Prevalence %)

Study by (Jackson and (Setasuban and (Kelly and Dent and Kelly, Davies and Blake and Collins et Moore and Arundel, Waddell, 1973) 2,b Ng, 1975) 3,b 1976) 3,a,b Nicholas, Overend, al ., O’Callaghan, 1971) 1,a 1977)3,a 1982) 1,b 1983) 3,c 1985) 4,d No. of dogs surveyed and 792, farm dogs 76, pound 464, pound 66, urban, pound; 110, farm dogs 734, pound 110, vet 1614, vet location 301, farm dogs clinic clinic

Helminth Ancylostoma spp. * 76.3 35.7 * * 2.1 * 3.0 Dipylidium caninum 17.4 * 67.5 13.6 10.0 57.0 * 0.6 Echinococcus granulosus 3.0 * - 4.1 7.2 - * * Spirometra erinacei 0.1 * 0.8 0.5 0.9 0.6 * * Strongyloides stercoralis * * 0.2 * * - * * Taenia spp. - * - - 5.4 - * 0.5 Taenia hydatigena 12.1 * - 11.2 13.6 3.0 * * Taenia ovis 2.6 * - 5.5 - 0.1 * * Taenia pisiformis 35.4 * 1.7 22.3 9.1 6.1 * * Taenia serialis 2.5 * 0.7 3.5 17.2 2.0 * * Toxascaris leonina * * 3.2 * * 4.0 * 1.4 Toxocara canis * * 35.1 * 3.6 37.8 * 6.4 Trichuris vulpis * * 60.6 * 0.9 41.4 * 8.8 Uncinaria stenocephala * - 9.5 * 5.4 25.9 * 4.1

Protozoa Cryptosporidium spp. * * * * * * * * Giardia duodenalis * * * * * * * * Isospora canis * * * * * 2.9 5.5 * Isospora ohionesis complex * * * * * 16.2 5.5 * Sarcocystis spp. * * * * * 26.3 20.9 *

67

Study by (Swan and (Collins et (Cornack and (Savini et al ., (Johnston and Milstein and Bugg et al ., 1999) 5,f Thompson, al ., 1097) 3,b O’Rourke, 1993) 5,f Gasser, Goldsmid, 1986) 5,f 1991) 2,e 1993) 1,e,g 1995) 6,f,g,h,i No. of dogs surveyed and 99, refuge; 131, 150, pound 112, sheep 67, household and 190, pound; 44, 55, refuge and 100, refuge; 100, pet shop; location breeder; 103, vet dogs vet clinics; 50, farm kennel; 107, vet clinic 77, vet clinic; 95, exercise clinic dogs; 15 refuge park; 152 vet areas; 49, breeding kennels dogs clinic

Helminth Ancylostoma spp. * * 20.1 - 6.7 1.8 1.9 Dipylidium caninum * * 4.1 - 0.8 - 0.2 Echinococcus granulosus * * * * * * * Spirometra erinacei * * - 1.5 0.2 * - Strongyloides stercoralis * * * * * * 0.2 Taenia spp. * * 4.5 - 0.8 - - Taenia hydatigena * * * * * * * Taenia ovis * * * * * * * Taenia pisiformis * * * * * * * Taenia serialis * * * * * * * Toxascaris leonina * * - - 0.2 - - Toxocara canis * * - - 9.5 10.9 1.7 Trichuris vulpis * * - 0.8 8.1 1.8 - Uncinaria stenocephala * * - 5.3 * - -

Protozoa Cryptosporidium spp. * * * * 11.0 1.8 - Giardia duodenalis 21.0 30.7 * 10.6 7.7 14.5 22.1 Isospora canis * * * 1.5 7.9 * 6.9 Isospora ohionesis complex * * * 2.3 7.9 * 4.5 Sarcocystis spp. * * * 31.1 19.5 * 6.2 (*) Not examined for; (-) not found. State/Territory in Australia: 1Victoria; 2Queensland; 3New South Wales; 4South Australia; 5Western Australia; 6Tasmania. a b c d e f g Techniques used in recovery of parasites: Arecoline purged; Necropsy; NaCl flotation; MgSO 4 flotation; NaNO 3 flotation; ZnSO 4 flotation; Modified Ziehl- Neelsen method; hFormalin-ethyl acetate sedimentation; iGiardia Ag CELISA

68

Table 3.2 Results of previous intestinal parasite studies in domestic cats in Australia (Prevalence %)

Study by (Kelly and (Pavlov and (Wilson-Hanson (Shaw et al ., (Collins et (Moore and (Swan and Ng, 1975) 2,a Howell, and Prescott, 1983) 4,a,f al ., 1983) 2,b O’Callaghan, Thompson, 1986)4,e 1977) 5,a 1982) 1,a 1985) 3,c No. of cats surveyed and 404, pound 42, feral; 58, 400, refuge 30, feral; 71, vet clinic 376, vet clinic 109, refuge; 45, location refuge 726, refuge breeder; 72 vet clinic

Helminth Aelurostrongylus abstrusus 11.4 19.0 3.8 - * - * Ancylostoma spp. 34.7 3.0 19.0 - * 0.8 * Capillaria spp. 5.2 - 6.0 - * - * Dipylidium caninum 39.4 29.0 19.0 32.9 * 1.3 * Spirometra erinacei 11.4 15.0 8.5 4.5 * - * Taenia spp. - - - - * 2.7 * Taenia serialis - - 0.3 - * * * Taenia taeniaeformis 6.9 25.0 - 9.9 * * * Toxascaris leonina 0.74 - 0.3 0.1 * 3.7 * Toxocara cati 57.2 25.0 24.5 11.8 * 5.3 * Uncinaria stenocephala 0.5 - - - * 0.3 *

Protozoa Cryptosporidium spp. * * - - - * * Giardia duodenalis * * - - - * 14.0 Isospora felis * * 10.5 1.1 4.2 * * Isospora rivolta * * 2.0 0.8 1.4 * * Sarcocystis spp. * * - - 1.4 * * Toxoplasma gondii * * 0.5 0.1 - * *

69

Study by (Collins et al ., 1987) 2,a (Sargent, 1997) 4,e (McGlade et al ., 2003)4,d,e,g,h No. of cats surveyed and 50, pound 162, refuge and vet 125, boarders; 120, refuge; 35, location clinic pet shop; 95, breeders; 43, private owners

Helminth Aelurostrongylus abstrusus * 0.6 0.2 Ancylostoma spp. * - 0.2 Capillaria spp. * - - Dipylidium caninum * - - Spirometra erinacei * - 0.5 Taenia spp. * - - Taenia serialis * - - Taenia taeniaeformis * - - Toxascaris leonina * - 2.2 Toxocara cati * 1.2 0.5 Uncinaria stenocephala * - -

Protozoa Cryptosporidium spp. * 1.2 10.0 (PCR) Giardia duodenalis 16.0 - 80.0 (PCR) Isospora felis * 12.9 4.4 Isospora rivolta * 6.1 1.4 Sarcocystis spp. * - - Toxoplasma gondii * 1.2 -

(*) Not examined for; (-) not found. State/Territory in Australia: 1Queensland; 2New South Wales; 3South Australia; 4Western Australia; 5Australian a b c d e Capital Territory. Techniques used in recovery of parasites: Necropsy; NaCl flotation; MgSO 4 flotation; NaNO 3 flotation; ZnSO 4 flotation; fSucrose flotation; gMalachite green staining; hPCR

70

3.2 Materials and Methods

Between March 2004 and September 2005 faecal samples were collected from dogs and cats from across Australia, from both urban and rural locations. A total of 1400 canine and 1063 feline faecal samples were collected from 59 veterinary clinics and

26 refuges: refuges (dogs n=590, cats n= 491), and dogs/cats presented to veterinary clinics without gastrointestinal (GI) complaints (dogs n=810, cats n=572). On the day of collection faecal samples were preserved in 10% formalin for microscopic screening. There was a time lag of up to 3 months before samples were sent to the laboratory. Once at the laboratory samples were refrigerated at 4 °C and the time taken to process the samples was highly variable, ranging from immediately to 6 months.

Demographic data (age, gender, neutering status, and breed) and anthelmintic history were collected for each animal sampled. A questionnaire was provided to pet owners to collect information on factors likely to impact specifically on the prevalence of GI parasites such as diet, number of dogs/cats in a household, time spent with other dogs/cats, time spent in an area used by other dogs/cats, predation of other animals by the pet, whether the pet spent time inside or outside the house/household, place of defaecation and how excrement was disposed of and frequency of disposal.

Initially it was anticipated that seasonal trends in parasitism would be investigated, unfortunately however, there was a great lag between when samples were collected and when they were sent to the laboratory for testing. Consequently, for many undated samples it was not possible to ascertain when they were collected.

71

The locations of the places sampled were categorised into one of three different climatic zones: tropical, arid or temperate (Figure 2.1). Most of the human and pet population in Australia is concentrated in two widely separated coastal regions: the south-east and east and the south-west of the continent. In both coastal regions the populations are concentrated in urban centres, particularly the State and Territory capital cities (Bureau of Meteorology website, 9/1/04). The locations sampled and proportions of samples collected for each location were dependent on the distribution of the human population in Australia (Figure 2.1). However, samples were also collected from isolated and less populated regions.

Formalised faecal samples were first examined macroscopically for the detection of proglottids and then screened using a standard sedimentation in water technique, a

Malachite Green stain for Cryptosporidium oocyts (Elliot et al ., 1999) followed by saturated salt and D-glucose centrifugal flotation and microscopy, as previously described by Henriksen and Christensen (1992).

Data were analysed and statistical comparisons were performed using SPSS (SPSS for

Windows, Version 14.0) and Excel 2002 (Microsoft).

Association between parasitism and host and management factors were initially made using univariate analyses of odds ratios and their 95% confidence intervals (Martin et al ., 1987), the Chi-square test for independence or continuous data were analysed using Kruskal Wallis or one-way analysis of variance (ANOVA), depending on the homogeneity of variance (Petrie and Watson, 1999). Multivariable logistic regression was then used to quantify the association between the presence of parasites with each variable after adjusting for other variables. Only variables significant at P < 0.25 in the univariable analysis were considered eligible for inclusion in the multiple logistic

72 regression analysis (Hosmer and Lemeshow, 1989; Frankena and Graat, 1997).

Dummy variables were generated for any categorical variable with more than two levels. Backward elimination was used to determine which factors could be dropped from the multivariable model (Hosmer and Lemeshow, 1989). The goodness of fit of the model was assessed with the Hosmer-Lemeshow statistic (Lemeshow and Hosmer,

1982).

3.3 Results

The overall prevalence of gastrointestinal parasites in dogs and cats was 23.9% (CI

21.7-26.1) and 18.4% (CI 16.1-20.7), respectively (Table 3.3 and 3.4). Of the dogs

19.8% (CI 17.7-22.0) were infected with a single species of parasite, and 4.1% (CI

3.1-5.1) with multiple species. Meanwhile, 15.7% of the cats (CI 13.5-18.0) were infected with a single species and 2.7% (CI 1.7-3.7) with multiple species. Protozoa were the most prevalent parasites for both dogs (16.1%, CI 14.2-18.0) and cats

(11.4%, CI 9.5-13.3). Overall Giardia was the most prevalent parasite in dogs (9.3%,

CI 7.8-10.8) followed by hookworm (6.7%, CI 5.4-8.0), while Isospora felis was the most prevalent parasite in cats (5.6%, CI 4.2-7.0), followed by Toxocara cati (3.2%,

CI 2.1-4.3).

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Table 3.3 The mean prevalence (%) of gastrointestinal parasites 1 of dogs from

refuge and veterinary clinics in Australia

Parasite Source of dog

Refuge (n= 590) Vet Clinic (n=810) Total (n= 1400)

Overall protozoa 22.0 (18.7-25.3) a 11.9 (9.7-14.1) 16.1 (14.2-18.0)

Overall helminth 15.9 (13.0-18.9) 4.9 (3.4-6.4) 9.6 (8.1-11.1)

Giardia spp. 14.4 (11.6-17.2) 5.5 (3.9-7.1) 9.3 (7.8-10.8)

Hookworm spp. 10.7 (8.2-13.2) 3.9 (2.6-5.2) 6.7 (5.4-8.0)

Sarcocystis spp. 3.2 (1.8-4.6) 3.9 (2.6-5.2) 3.6 (2.6-4.6)

Isospora ohioensis 5.6 (3.7-7.5) 2.0 (1.0-3.0) 3.5 (2.5-4.5) complex

Trichuris vulpis 3.1 (1.7-4.5) 0.9 (0.3-1.6) 1.8 (1.1-2.5)

Toxocara canis 2.4 (1.2-3.6) 0.4 (0-0.8) 1.2 (0.6-1.8)

Isospora canis 1.4 (0.5-2.3) 0.9 (0.3-1.6) 1.1 (0.6-1.7)

Cryptosporidium 0.7 (0.03-1.4) 0.5 (0.01-1.0) 0.6 (0.2-1.0) spp.

Toxascaris leonina 0 0.2 (0-0.5) 0.1 (0-0.3)

Spirometra erinacei 0.2 (0-0.6) 0.1 (0-0.3) 0.1 (0-0.3)

Dipylidium caninum 0.3 (0-0.7) 0 0.1 (0-0.3)

Total parasite 34.6 (30.8-38.4) 16.0 (13.5-18.5) 23.9 (21.7-26.1) infection a 95% confidence intervals

1Determined from faecal samples see text for methodology

Note: some dogs were infected with more than one parasite

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Table 3.4 The mean prevalence (%) of gastrointestinal parasites 1 of cats from

refuge and veterinary clinics in Australia

Parasite Source of cat

Refuge (n= 491) Vet Clinic Total (n= 1063)

(n=572)

Overall protozoa 19.8 (16.3-23.3) a 4.2 (2.6-5.8) 11.4 (9.5-13.3)

Overall helminth 12.0 (9.0-14.9) 4.4 (2.7-5.8) 7.9 (6.3-9.5)

Isospora felis 10.2 (7.5-12.9) 1.7 (0.6-2.8) 5.6 (4.2-7.0)

Toxocara cati 4.9 (3.0-6.8) 1.7 (0.6-2.8) 3.2 (2.1-4.3)

Isospora rivolta 5.5 (3.5-7.5) 0.3 (0-0.7) 2.7 (1.7-3.7)

Spirometra erinacei 3.5 (1.9-5.1) 2.1 (0.9-3.3) 2.7 (1.7-3.7)

Cryptosporidium 3.5 (1.9-5.1) 1.0 (0.2-1.8) 2.4 (1.3-3.1)

Giardia spp. 2.6 (1.2-4.0) 1.4 (0.4-2.4) 2.0 (1.2-2.8)

Hookworm spp. 2.9 (1.4-4.4) 0.2 (0-0.6) 1.4 (0.7-2.1)

Aelurostrongylus 1.8 (0.6-3.0) 0 0.8 (0.3-1.3) abstrusus

Toxascaris leonina 0.2 (0-0.6) 1.7 (0.6-2.8) 0.3 (0-0.6)

Dipylidium caninum 0.4 (0-1.0) 0 0.2 (0-0.5)

Capillaria aerophila 0.2 (0-0.6) 0 0.1 (0-0.3)

Toxoplasma/Hammondia 0.2 (0-0.6) 0 0.1 (0-0.3)

Total parasite infection 30.3 (26.2-34.4) 8.2 (6.0-10.4) 18.4 (16.1-20.7) a 95% confidence intervals

1Determined from faecal samples see text for methodology

Note: some cats were infected with more than one parasite

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Of the dogs from the refuges, 31.3% had received at least one anthelmintic treatment during their time in the shelter, and of the owned dogs from the veterinary clinics

81.2% had received at least one anthelmintic treatment in the previous 12 months. Of the treated dogs from the veterinary clinics 98.8% had been treated with an

“allwormer” (broad spectrum anthelmintic). The prevalence of helminths in dogs from refuges which were not treated with an anthelmintic was 19.6%, and in treated dogs was 9.7%. In dogs from veterinary clinics, the prevalence of helminth infection was

7.2% and 4.3% in non-treated and treated dogs, respectively.

Similarly, 41.1% of refuge cats had received at least one anthelmintic treatment, while

80.0% of owned cats had received at least one anthelmintic treatment in the previous

12 months. Of the owned cats which had been treated, 86.7% had been treated with a broad spectrum anthelmintic. Cats from refuges which had not been treated with an anthelmintic had a helminth prevalence of 15.5%, compared to a prevalence of 7.4% in treated cats. The prevalence of helminth infection in cats which had not been treated from veterinary clinics was 9.2% compared to 3.1% in treated cats.

Dogs were most likely to be parasitised if they were from a refuge and less than/equal to one year of age; the same factors existed for infection with a protozoan parasite

(Table 5). Dogs were at most risk of helminth infection if they were from a refuge, in an urban location, in a tropical climate, less than one year of age, and had not received any anthelmintic treatment in the previous 12 months (Table 3.5).

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Table 3.5 The results of the multiple logistic regression model for relationships

between dog host variables and parasitism (odds ratios are relative to the

opposite category: refuge vs veterinary clinic, young versus older than 1 year,

tropical vs not a tropical climate, urban vs rural, received anthelmintic vs none)

Factor Association with infection P-value Constant

odds ratio (95% CI)

Parasitic infection -1.029

Refuge 2.7 (2.0-3.6) <0.0001

< 1 year of age 2.8 (2.1-3.7) <0.0001

Protozoan infection -1.758

Refuge 2.0 (1.5-2.7) <0.0001

< 1 year of age 3.2 (2.3-4.3) <0.0001

Helminth infection -2.003

Refuge 2.5 (1.3-5.0) 0.005

Tropical climate 4.2 (2.5-7.1) <0.0001

From urban location 2.5 (1.7-5.0) <0.0001

< 1 year of age 1.6 (1.0-2.8) 0.06

Received no 2.0 (1.3-5.0) 0.01 anthelmintics

Dogs from veterinary clinics were most likely to be infected with a parasite if they were less than/equal to one year of age, spent time in the backyard, and the owner did not pick up the dog’s faeces at least once a week (Table 3.6). Protozoan infection was more likely in a dog from a veterinary clinic which was less than/equal to one year of age (Table 3.6). Dogs from veterinary clinics were more at risk of helminth infection if they were less than/ equal to one year of age, from a rural location, the dog’s owner

77 did not pick up the dog’s faeces at least once a week, and the dog spent time in the backyard (Table 3.6).

Table 3.6 The results of the multiple logistic regression model for relationships between host and management variables and parasitism for dogs from veterinary

clinics (odds ratios are relative to the opposite category: young versus older than

1 year, spends time outside house vs majority of time spent inside, owner pick’s

up feaces weekly vs not so, urban vs rural)

Factor Association with P-value Constant

infection odds ratio

(95% CI)

Parasitic infection -3.509

< 1 year of age 4.1 (1.6-10.2) 0.003

Spends time outside house 7.8 (1.0-63.8) 0.055

Owner does not pick up dog’s 2.8 (1.1-7.1) 0.032 faeces weekly

Protozoan infection -3.495

< 1 year of age 3.4 (2.2-5.3) <0.0001

Helminth infection -3.105

< 1 year of age 2.3 (1.2-4.5) 0.014

Rural 2.0 (1.0-3.3) 0.056

Owner does not pick up dog’s 3.3 (1.7-5.0) 0.001 faeces weekly

Spends time outside house 3.2 (1.1-9.2) 0.031 (in backyard)

Cats were at most risk of a parasitic infection, and infection with a protozoan parasite, if they were from a refuge and less than/equal to one year of age (Table 3.7). Helminth

78 infection was more common in those cats originating from a refuge, and cats which had not received any anthelmintics in the last 12 months (Table 3.7).

Table 3.7 The results of the multiple logistic regression model for relationships between cat host variables and parasitism (odds ratios are relative to the opposite

category: refuge vs veterinary clinic, young versus older than 1 year, received

anthelmintic vs none)

Factor Association with infection P-value Constant

odds ratio (95% CI)

Parasitic infection -0.595

Refuge 4.2 (2.9-6.1) <0.0001

< 1 year of age 1.6 (1.2-2.3) 0.006

Protozoan infection -1.0

Refuge 4.6 (2.9-7.4) <0.0001

< 1 year of age 2.4 (1.6-3.6) <0.0001

Helminth infection -1.676

Refuge 2.0 (1.1-3.4) 0.015

Received no 2.8 (1.6-4.8) <0.0001 anthelmintics

Parasite infection in cats from veterinary clinics was more common in those cats less than/equal to one year of age, and cats which had regular contact with other cats

(Table 3.8). Veterinary clinic cats were more likely to be infected with a protozoan parasite if they were less than/equal to one year of age (Table 3.8). Risk factors for helminth infection included: spending time outside the house and not receiving any anthelmintic treatments in the previous 12 months (Table 3.8).

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Table 3.8 The results of the multiple logistic regression model for relationships

between host and management variables and parasitism for cats from veterinary

clinics (odds ratios are relative to the opposite category: young versus older than

1 year, regular contact with other cats vs non- regular contact, spends time

outside house vs majority of time spent inside, received anthelmintic vs none)

Factor Association with infection P-value Constant

odds ratio (95% CI)

Parasitic infection -2.293

< 1 year of age 2.1 (1.0-4.1) 0.042

Regular contact with other 1.9 (0.9-3.7) 0.076 cats (cats which do not belong to the household)

Protozoan infection -2.231

< 1 year of age 5.46 (2.1-14.1) <0.0001

Helminth infection -2.669

Spends time outside house 3.4 (1.3-8.7) 0.012

Received no 4.0 (1.5-10.5) 0.005 anthelmintics

3.4 Discussion

From the results it is evident that protozoan infection is more common in dogs and cats than helminth infection in Australia. The overall prevalence of helminth infection in dogs and cats from veterinary clinics was low, and most pet owners had treated their pet with an anthelmintic. The prevalence of helminth infection in dogs and cats which had not received anthelmintic treatment and which were sampled at veterinary clinics was also reasonably low. It should be mentioned that given that microscopy

80 was used to identify parasite positive animals, this study is likely to underestimate the true prevalence of parasites. Yet even in consideration of this, it would seem that many well cared for dogs and cats receiving anthelmintic treatments have extremely low parasitic burdens or are, in fact, free from helminth infection.

Giardia was found to be the most prevalent parasite in dogs. Although none of the dogs sampled exhibited any overt clinical signs, there is a growing body of evidence to suggest that asymptomatic infections with Giardia may still be associated with nutritional disease (Buret et al ., 2002a; Buret et al ., 2002b; Chin et al ., 2002; Scott et al ., 2002). Recent evidence suggests that products released by the parasite can alter the gut epithelial permeability through a direct cytopathic effect (Chin et al ., 2002;

Scott et al ., 2002). Furthermore, dogs and cats can harbour infections with either zoonotic or host specific assemblages of Giardia (Caccio et al ., 2005; Leonhard et al .,

2007). We believe that further research is needed to investigate the zoonotic potential of this parasite and how pathogenic it really is.

There were no samples found positive for Taenia spp. or Echinococcus granulosus both of which are known to occur in Australia. Although faecal flotation is not a desirable method of detecting such parasites, the reason for their absence is most likely as a consequence of the dogs sampled in this study. Infection with these parasites is dependent on the dog’s access to the carcasses of rabbits, sheep, cattle, pigs, goats and macropods, which is an unlikely event for many pets in Australia.

However, E. granulosus remains of great concern in the focal hyperendemic regions of country Australia, where it is known to occur. The domestic dogs which tend to be infected with this parasite are farm working dogs (Jenkins et al ., 2006) and are unlikely to be taken to veterinary clinics routinely, or dogs allowed to free range that

81 may scavenge on carcasses of infected macropods in endemic areas. Implementation of a targeted educational program for the owner’s of such dogs is of the greatest necessity (Jenkins et al ., 2006). In addition, there is growing awareness of the emerging public health risks associated with the encroachment of infected wildlife into urban areas close to a number of Australian cities, and spillover to domestic dogs as a consequence of human activity (Thompson, 2001; Jenkins, 2006 ).

Not surprisingly, parasitic infection in both dogs and cats was more frequent in refuge and young animals. The animals from the refuges were less likely to have been treated with an anthelmintic, and may have also been more prone to infection within the refuge due to the immunosuppressive effects of stress, direct contact with many other animals and their excrement, and environmental contamination. Higher parasite prevalences often occur in young animals due to their immature immune systems, and the transmammary and transplacental routes of infection of some parasites, namely

Ancylostoma caninum and Toxocara spp. (Bowman et al ., 2003).

Hookworm were the most common helminth infection in dogs, and dogs originating from a tropical climate were more at risk than dogs which were from other areas. The species of hookworm determined during this study, their distribution and the effect of climate have been discussed elsewhere (Palmer et al ., 2007). The importance of picking up pets’ faeces regularly in the backyard, in an effort to prevent the larval development of helminths to an infective stage, was highlighted by the results of the present study.

Regular contact with other cats was a risk factor for parasitism in owned cats. If a cat had access to other cats outside the household, one could assume they would also have

82 access to the faeces of these other animals. Hence, they would be at risk of exposure to those parasites which have and can have, a direct life cycle, including Isospora ,

Giardia , Cryptosporidium , Toxocara , Toxascaris and hookworm. Owned cats were also at more risk of helminth infection if they spent time outside the household, again not only increasing the potential of exposure to a contaminated environment, but also to paratenic hosts, especially given that Spirometra erinacei was the most prevalent parasite in owned cats.

In conclusion the prevalence of intestinal parasites in Australian pet dogs and cats is low, suggesting that the frequent administration of broad spectrum anthelmintics has had a significant impact on the epidemiology of gastrointestinal parasitism in domestic dogs and cats. Consideration should now be given to their use in the future, and in this respect, it is essential that anthelmintics are administered prophylactically to animals less than 6 months of age, given that it is well established that this demographic is at high risk of infection. In older animals a more flexible approach may be warranted on advice from a veterinarian. Prophylactic administration of anthelmintics in pets residing in a tropical climate and in environments where the risk of infection is greater such as breeding kennels, should also be considered given the risk of hookworm infection (Palmer et al ., 2007).

Most of the dogs and cats which were parasitised were infected with a single species of parasite, yet the majority of anthelmintics used in pets in Australia are broad spectrum. Faecal examination prior to anthelmintic treatment would enable the targeted treatment of parasites, with the clinician selecting an anthelmintic which had spectrum against either nematodes or cestodes. An important consideration for drug

83 selection is the recent findings of Kopp et al . (2007) were a high-level of pyrantel resistance was found in Australian isolates of A. caninum .

Although Giardia infected animals are frequently asymptomatic, treatment is necessary if this parasite is recovered, given the zoonotic potential and because the significance of infection is not completely understood. Products containing febantel are effective at stopping the shedding of Giardia cysts (Barr and Bowman, 1994;

Meyer, 1998; Zajac et al ., 1998), and are now available for use in dogs.

Finally, in order to prevent an increase in the prevalence of pet parasite infection and zoonoses, it is imperative that veterinarians continue to educate their clients on the importance of good husbandry practices and correct anthelmintic and antiprotozoal drug administration. Emphasis on client education as a means of parasite prevention should also be instilled during veterinary training.

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Chapter 4

The veterinary and public health significance of hookworm

in dogs and cats in Australia and the status of A. ceylanicum

This chapter is a published paper: PALMER, C. S., TRAUB, R. J., ROBERTSON, I.

D., HOBBS, R. P., ELLIOT, A., WHILE, L., REES, R. & THOMPSON, R. C. (2007)

The veterinary and public health significance of hookworm in dogs and cats in

Australia and the status of A. ceylanicum . Veterinary Parasitology, 145 , 304-13.

Please note that this paper was published prior to the completion of the study due to the urgency of reporting the discovery of A. ceylanicum in Australia. Opportunistic faecal samples collected from dogs from Aboriginal Communities were also included in this paper. As a consequence the prevalences reported in this chapter are slightly different to those reported in the previous chapter.

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4.1 Introduction

Hookworm infection in dogs and cats can result in serious disease and even death.

Furthermore, most of the species of hookworms which infect dogs and cats are zoonotic. There are no current data available on the prevalence of hookworm in

Australia, possibly because of a sense of security provided by the widespread use of broad spectrum anthelmintics. Indiscriminant use of anthelmintics without knowledge of the prevalence of the parasite in question may hasten the development of anthelmintic resistance (Thompson, 1999b; Cohen, 2000; Laffont et al ., 2001;

Thompson and Roberts, 2001; Irwin, 2002). Moreover, knowledge of the prevalence of hookworm is important with regard to public health. Previous studies in Australia have implicated Ancylostoma caninum as the leading cause of human eosinophilic enteritis (EE) (Prociv and Croese, 1990). Between 1988 and 1992 an outbreak totalling 150 cases of EE was reported and the dog hookworm A. caninum implicated as the causal agent (Loukas et al ., 1992; Croese et al ., 1994).

Traditionally, identification of hookworm species in dogs and cats required coprological or post-mortem examination of the adult worms, based on morphological differences between the species of Ancylostoma (Yoshida et al ., 1971; Soulsby, 1982).

This is very time consuming, labor intensive and requires skilled personnel (Traub et al ., 2004b). In some instances, the species of hookworm involved in an infection has been identified based on the morphology of the eggs recovered from the faeces

(Dunsmore and Shaw, 1990), but this is not appropriate as there is overlap in egg dimensions between most species of hookworm affecting dogs and cats. However,

Traub et al . (2004b) recently developed a highly species-specific and sensitive PCR-

RFLP technique to detect and differentiate canine Ancylostoma spp. directly from eggs in faeces, this obviates the requirement for the use of tedious diagnostic methods

86 of hookworm identification. This technique was applied to a large scale epidemiological study and allowed canine hookworm identification to be conducted rapidly, with ease and accuracy (Traub et al ., 2004b).

The hookworm species previously thought to have been identified in Australian dogs and cats included A. caninum , A. tubaeforme , A. braziliense , A. ceylanicum and

Uncinaria stenocephala . However, the only reports of A. ceylanicum in Australia

(Stewart, 1994, Gasser et al ., 1996, Adams, 2004) have been found to be based on a misidentification of A. braziliense as A. ceylanicum (E Silva et al ., 2006; Traub et al .,

2007).

There is no current data on the prevalence of hookworm in Australia. High hookworm prevalence was recorded in surveys conducted over twenty years ago (Table 4.1).

Ancylostoma caninum was recorded as the predominant hookworm of dogs in the warmer regions of Australia. Although A. caninum is regarded as an uncommon parasite of cats (Setasuban and Waddell, 1973; Kelly and Ng, 1975; Wilson-Hanson and Prescott, 1982; Beveridge, 2002), Stewart (1994) found it in 6 of 16 cats examined in Townsville. Uncinaria stenocephala is a common parasite of dogs in southern Australia and is less common in cats (Beveridge, 2002).

The predominant species of hookworm in cats in Australia is considered to be A. tubaeforme (Table 4.1) .

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Table 4.1 Previous prevalence studies in Australia ordered chronological

Location Source of animals Method of diagnosis Dog/ cat Species of Prevalence Reference hookworm (%) Cairns Pound Necropsy Dog (n = 10) A. caninum 100 Setasuban and Waddell (1973) Brisbane Pound Necropsy Dog (n = 66) A. caninum 67 Sydney Pound Necropsy Dog (n = 464) A. caninum 36 Kelly and Ng (1975) North-eastern Pound Necropsy Dog (n = 734) A. caninum 2 Blake and Overend (1982) Victoria Centeral western Sheep dogs Faecal exam Dog (n = 112) A. caninum a 20 Cornack and O’Rourke (1991) Queensland South of Sydney Aborignial Community Necropsy Dog (n = 15) A. caninum 100 Jenkins and Andrew (1993) Kimberly Aboriginal Community Necropsy Dog (n= 188) A. caninum 51 Meloni et al ., (1993) Perth Vet practices, exercise areas, pet Faecal exam Dog (n = 421) A. caninum a 2 Bugg et al ., (1999) shops, refuges, breeding kennels Sydney Pound Necropsy Cat (n = 404) A. tubaeforme 35 Kelly and Ng (1975) Brisbane Pound Necropsy Cat (n = 400) A. tubaeforme 19 Wilson-Hanson and Prescott (1982) Brisbane Pound Necropsy Cat A. tubaeforme 81 Prescott (1984) Kimberly Aboriginal Community Necrosy Cat (n = 34) A. tubaeforme 20 Meloni et al ., (1993) Northern Territory Feral cats Necropsy Cat A. tubaeforme 13 O’Callaghan and Beveridge (1996) Sydney Pound Necropsy Dog (n = 464) U. stenocephala 9.5 Kelly and Ng (1975) Cat (n = 404) U. stenocephala 0.5 Tasmania Feral cats Necropsy Cat (n = 86) U. stenocephala 2 Gregory and Munday (1976) North eastern Pound Necropsy Dog (n = 734) U. stenocephala 26 Blake and Overend (1982) Victoria Adelaide Vet practices Faecal exam Dog (n = 1614) U. 4 Moore and O’Callaghan (1985) stenocephala a Cat (n = 376) U. 0.3 stenocephala a Perth Private owners, refuges, boarding Faecal exam Cat (n = 418) Unknown 0.2 McGlade et al ., (2003) cats, pet shops, breeders a Diagnosis of hookworm species was made according to egg measurements which is not a reliable diagnostic method

88

Prior to 1951, A. ceylanicum was regarded as a synonym of A. braziliense (Yorke &

Maplestone, 1926; Skrjabin et al ., 1951), until Biocca (1951) provided morphological evidence that they were different. Ancylostoma braziliense has only been identified in a handful of studies and has been reported in dogs and cats in north Queensland

(Heydon, 1929; Seddon, 1958; Heydon and Bearup, 1963; Setasuban and Waddell,

1973; Taveros, 1990; Stewart, 1994) with a single occurrence in a dog from Sydney

(Kelly and Ng, 1975). Specimens recovered by Heydon (1929) from cats in

Townsville were identified as A. braziliense prior to the morphological criteria established by Biocca (1951), and hence need to be reexamined.

The primary aim of this study was to establish the prevalence, species distribution and risk factors associated with hookworm infection in dogs and cats in Australia and in doing so, readdress the veterinary and public health concerns associated with these parasites as well as their control. The PCR-RFLP designed by Traub et al . (2004b) was modified to allow differentiation of all hookworm species known to infect dogs and cats and was applied in the epidemiological screening of faecal samples. Material from the studies of Heydon (1929), identified as A. braziliense , were re-examined.

4.2 Material and Methods

4.2.1 Source of faeces

Between March 2004 and September 2005 faecal samples were collected from dogs and cats from across Australia from both urban and rural locations. A total of 1391 canine faecal samples were collected from 3 sources: refuges (n=568), dogs presented to veterinary clinics without gastrointestinal (GI) complaints (n=766) and Aboriginal communities (n=57). A total of 1027 faecal samples were collected from cats from 2

89 sources: refuges (n=461) and cats presented to veterinary clinics without GI complaints (n=566).

Demographic data (age, gender, neutering status, and breed) and worming history were collected for all animals sampled. A questionnaire was administered to pet owners to collect information on factors likely to impact specifically on the prevalence of GI parasites such as diet, number of dogs in a household, number of cats in a household, time spent with other dogs and cats, time spent in an area used by other dogs and cats, place of defaecation and how excrement was disposed of.

The locations of the places sampled were categorised into one of three different climatic zones (tropical, arid or temperate) (Fig. 2.1). These three climatic zones were a simplification of the six climatic zones depicted on a climatic map for temperature and humidity produced by the Bureau of Meteorology (Bureau of Meteorology website, 9/1/04). According to the present study a ‘tropical’ climate included those regions on the climatic map which were classified as having a hot humid summer or a warm humid summer, while an ‘arid’ climate included areas which were deemed as having a hot dry summer, mild winter or hot dry summer, cold winter and a

‘temperate’ climate included those areas classified as warm summer, cool winter or mild/warm summer, cold winter.

4.2.2 Parasitological procedures

Formalised faecal samples were examined for parasites initially using a simple faecal smear followed by centrifugal flotation in saturated sodium chloride and glucose, and microscopy, as previously described by Henriksen and Christensen (1992).

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4.2.3 Molecular methods

One hundred and ten samples, from 96 dogs and 14 cats, which were found positive for hookworm via microscopy, were then characterised using molecular methods.

4.2.3.1 DNA extraction

Two hundred micrograms of canine faeces was suspended in 1.4 ml ATL tissue lysis buffer (Qiagen, Hilden, Germany). This suspension was then subjected to five cycles of freeze-thawing at liquid nitrogen temperatures followed by boiling for 10 minutes.

DNA was then isolated from the supernatant using the QIamp DNA Mini Stool Kit

(Qiagen, Hilden, Germany) according to manufacturer’s instructions. Final elutions of

DNA were made in 50 µl of elution buffer instead of 200 µl as recommended by the manufacturer.

4.2.3.2 PCR for differentiating A. caninum, A. tubaeforme, A. ceylanicum and

Uncinaria stenocephala from A. braziliense

A section of the internal transcribed spacer (ITS) -1, 5.8S and ITS-2 regions of A. caninum , A. tubaeforme , A. ceylanicum , A. braziliense and U. stenocephala was amplified using previously published primers, PCR reaction, and cycling conditions

(Traub et al ., 2004b). An amendment to this PCR was however recently made by

Traub et al. (2007) whereby it was recognised that the reference sequence of A. ceylanicum was in fact that of A. braziliense . Hence RTGHFI (5’-

CGTGCTAGTCTTCAGGACTTTG-3’) and RTABCR1 (5’-

CGGGAATTGCTATAAGCAAGTGC-3’) were used to amplify a 545 bp region of A. caninum , A. tubaeforme , A. ceylanicum and U. stenocephala . In a separate PCR a 673 bp region of A. braziliense was amplified using RTGHF1 and the highly specific

91 reverse primer RTAYR1 (5’-CTGCTGAAAAGTCCTCAAGTCC-3’) (Traub et al .,

2004b).

4.2.3.3 PCR-linked restriction fragment length polymorphism (RFLP)

Appropriate restriction enzymes for the PCR-RFLP to differentiate the species of hookworms were selected using the PCR-RFLP MONSTER © (www.rflpmonster.com) program. PCR-RFLP Monster © is a web-based PCR-RFLP assay design program. It replaces the manual and laborious task of choosing the most suitable restriction endonuclease or combination of endonucleases to distinguish a given set of organisms.

In accordance with the outputs produced by PCR-RFLP Monster ©, amplified ITS PCR products of RTGHF1-RTABCR1 were subjected to direct digestion with HinF1 in order to differentiate A. caninum and A. tubaeforme from A. ceylanicum and U. stenocephala (Table 4.2). Restriction enzyme RSaI was then used to differentiate U. stenocephala and A. tubaeforme , while A. caninum and A. ceylanicum have identical cutting patterns with this enzyme (Table 4.2). The RFLP reaction for HinFI (Promega) and RSaI (Promega) restriction endonuclease were identical. Ten microlitres of PCR product were digested with 0.5 µl (5 units) of a restriction endonuclease at 37ºC for

16h in a volume of 20 µl.

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Table 4.2 Restriction profile

Enzyme Species Band sizes

A. tubaeforme 7*, 50*, 85*, 187, 216

A. caninum 7*, 49*, 85*, 187, 216

HinFI A. ceylanicum 49*, 194, 301

U. stenocephala 40*, 194, 303

A. tubaeforme † 40*, 77*, 151, 277

40*, 77*, 428

RSaI A. caninum 268, 276

A. ceylanicum 286, 276

U. stenocephala 545

* Product too small to view

† Allelic polymorphism produces two separate RFLP banding patterns

4.2.3.4 Sequencing of A. ceylanicum positive samples

Ten out of the 96 canine positive hookworm samples were identified as A. ceylanicum from the results of the RFLP. Four of these samples were reamplified using the aforementioned PCR and the PCR products were purified using Qiagen spin columns

(Qiagen) and sequenced using an ABI 3730 48 capillary DNA sequencer (Applied

Biosystems) using Big Dye version 3.1 terminators. Sequences were analysed using

Finch TV Version 1.3.1 (Geospiza Inc.) and were deposited to Genbank (Genbank accession numbers: DQ831518, DQ831519, DQ831517 and DQ831520). Sequences were then compared with A. ceylanicum (Genbank accession number DQ381541), two

93 of the sequences showed 100% identity (DQ831518 and DQ831519), while the other two (DQ831517 and DQ831520) were 99.8% and 99.6% similar respectively.

4.2.4 Morphological identification of specimens from the Queensland Museum

Specimens collected from cats in Townsville Australia, by GM Heydon between 1923 and 1928, stored in 70% ethanol at the Queensland Museum as A. braziliense under the Accession Numbers GL11582, GL11583 and GL11633, were identified morphologically. Identification was based on the structure of the teeth in the cutting plates, and the structure of the male bursal rays after Biocca (1951).

4.2.5 Statistical analysis

The prevalence and 95% confidence intervals (CI) were calculated for hookworm in both dogs and cats. Association between parasitism and host and management factors were initially made using univariate analyses of odds ratios and their 95% confidence intervals (Martin et al ., 1987), the Chi-square test for independence or the analysis of variance (Martin et al ., 1987). Associations between host factors (age, gender, neutering status and purebred/crossbred), management factors and climate and hookworm infection were evaluated for all dogs and cats sampled.

Multivariable logistic regression was then used where data were substantial enough to quantify the association between the presence of hookworm infection and host and management variables after adjusting for other variables. Only variables significant at

P < 0.25 in the univariate analysis were considered eligible for inclusion in the multiple logistic regression analysis (Hosmer and Lemeshow, 1989; Frankena and

Graat, 1997). Dummy variables were generated for any categorical variable with more than two levels. Backward elimination was used to determine which factors could be

94 dropped from the multivariable model (Hosmer and Lemeshow, 1989). The goodness of fit of the model was assessed with the Hosmer-Lemeshow statistic (Lemeshow and

Hosmer, 1982).

Data were analysed and statistical comparisons were performed using SPSS (SPSS for

Windows, Version 14.0) and Excel 2002 (Microsoft).

4.3 Results

4.3.1 Prevalence of hookworm

The overall prevalence of infection with hookworm determined by microscopy was

6.9% (95% confidence interval [CI] 5.6, 8.2) and 1.4% (95% CI 0.7, 2.1) for dogs and cats respectively. Dogs sourced from Aboriginal communities had the highest prevalence (14.0%) followed by those from refuges (11.4%) and from veterinary clinics (3.0%). Cats sourced from refuges had a higher prevalence (2.8%) than cats from veterinary clinics (0.2%). The prevalence of hookworm also varied greatly between geographical regions as shown in Table 4.3. The categorisation of prevalence according to state can however be misleading, as state boundaries are merely superimposed arbitrary delineations. The variation in hookworm prevalences is best correlated with climatic zones as reflected in Table 4.4.

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Table 4.3 Prevalence of hookworm in dogs according to the state in Australia

State Number of Total number of Prevalence

dogs positive dogs examined (%)

Northern Territory 23 131 17.5

Queensland 28 274 10.2

Western Australia 26 321 8.1

Victoria 6 152 3.9

New South Wales 8 219 3.7

Australian Capital Territory 2 85 2.3

Tasmania 2 100 2.0

South Australia 1 109 0.9

(Note: the prevalences of hookworm within a state tended to vary between the locations sampled, especially in those states which cover more than one climatic zone)

Table 4.4 Prevalence of hookworm in dogs according to climate

Climate Prevalence (%)

Tropical 17.3 (n=412)

Arid 1.7 (n=120)

Temperate 2.7 (n=859)

4.3.2 PCR-RFLP

Of the 96 microscopy positive dog hookworm samples 92 were amplified successfully by PCR. Of the 14 microscopy positive cat hookworm samples ten were amplified successfully. The inability to amplify all of the samples was thought to be associated with inhibitory factors within the PCR reaction or an inadequate amount of DNA. No

96 products were observed for the A. braziliense species specific PCR. PCR patterns of the hookworms amplified are shown in Fig 4.1.

Figure 4.1 PCR of the ITS of all of the canine and feline hookworm species. From left to right, lane 1 displays a 100 base pair molecular marker, lanes 2, 3, 4,5,6,7 and 8 display PCR amplified products of ITS regions of A. caninum , A. tubaeforme , A. ceylanicum U. stenocephala , mixed infection with A. caninum and

A. ceylanicum and mixed infection with A. caninum and U. stenocephala respectively (545bp), lane 8 displays PCR product of A. braziliense (673bp) and lane 9 is a negative control.

PCR products of RTGHF1-RTABCR1 were then further subjected to the RFLP using

HinFI and RSaI (Figs. 4.2 and 4.3). Of the 92 dog samples, 70.7% were found positive for single hookworm infections with A. caninum , 6.5% positive for single infections with A. ceylanicum , 10.9% positive for single infections with U. stenocephala , 4.3% had mixed infections with both A. caninum and A. ceylanicum and 2.2% had mixed infections with both A. caninum and U. stenocephala . All ten cat samples amplified had single infections; 70% were found positive for A. tubaeforme and 30% were found positive for A. caninum . The distributions of the different hookworm species identified are depicted in Tables 4.5 and 4.6.

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Figure 4.2 RFLP of the PCR product RTGHF1-RTABCR1 following digestion with restriction endonuclease HinFI. From left to right, the gel shows representatives of digested product of A. caninum in lane 2, digested product of

A. tubaeforme in lane 3, digested product of A. ceylanicum in lane 4, digested product of U. stenocephala in lane 5, digested product of a mixed infection with

A. caninum and A. ceylanicum in lane 6 and digested product of a mixed infection with A. caninum and U. stenocephala in lane 7.

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Figure 4.3 RFLP of the PCR product of RTGHF1-RTABCR1 following digestion with restriction endonuclease RSaI. From left to right, the gel show representatives of undigested U. stenocephala in lane 2, digested product of A. caninum in lane 3, digested product of A. tubaeforme in lane 4, digestive product of A. ceylanicum in lane 5, digested product of a mixed infection with A. caninum and A. ceylanicum in lane 6 and digested and undigested product of a mixed infection with A. caninum and U. stenocephala in lane 7.

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Table 4.5 Location of A. caninum, A. ceylanicum and U. stenocephala positive

samples in dogs

Species of hookworm Location of samples

Ancylostoma caninum Brisbane, Broome, Cairns, Darwin, Derby, Gapuwiyak,

Geraldton, Gold Coast, Sunshine Coast,

Hobart, Melbourne, Taree, Alice Springs

A. ceylanicum Broome, Brisbane, Sunshine Coast, Melbourne, Alice

Springs

Uncinaria Ballarat, Canberra, Geraldton, Melbourne, Moss Vale, stenocephala Perth, Port Lincoln, Sale, Sydney

Table 4.6 Location of A. tubaeforme and A. caninum positive samples in cats

Species of hookworm Location of samples

A. caninum Darwin, Alice Springs

A. tubaeforme Cairns, Brisbane, Bundaberg, Darwin

4.3.3 Morphological identification of specimens from the Queensland Museum

Three species of Ancylostoma were identified from the Museum specimens labelled as

A. braziliense . While A. braziliense was present in all 3 specimen containers, a single male specimen of A. ceylanicum was also present in GL11582 (Fig 4.4), and 24 male

A. tubaeforme in GL11633. This represents the first confirmed report of A. ceylanicum in Australia.

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Figure 4.4 Lateral view of male bursa of A. ceylanicum from a cat from

Townsville, Australia, collected by GM Heydon in 1926 (Queensland Museum

GL 11582). Scalebar 50µm.

4.3.4 Risk factors for parasitism

4.3.4.1 Multivariable analysis for dogs

All variables significant at P ≤ 0.25 on univariable analysis were included in the initial multivariable model. Dogs from refuges, dogs originating from a tropical climatic zone, dogs aged one year or less, and those dogs which had not received anthelmintics were significant in the final model for parasitism (Table 4.7).

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Table 4.7 The results of the multiple logistic regression model for the

relationships between the variables examined and hookworm infection in dogs

Factor odds ratio (95% CI) P-value Constant

-2.703

Dogs from refuges 2.9 (1.6-5.3) 0.001

Dogs not from a refuge 1.0

Dogs residing in a tropical climate 5.6 (3.3-9.5) <0.001

Dogs not from a tropical climate 1.0

Dogs ≤ 1 year of age 1.7 (1.0-2.9) 0.044

Dogs > 1 year of age 1.0

Dogs which had not received 2.6 (1.4-4.7) 0.002 anthelmintics treatment

Dogs which had received 1.0 anthelmintic treatment

4.3.4.2 Univariable analysis for dogs from veterinary clinics

There were too few dogs sourced from veterinary clinics positive for hookworm and as such only univariate analysis was conducted. Variables were considered significant if the P- value was ≤ 0.05. According to univariate analysis dogs from veterinary clinics were more likely to be infected with hookworm if they originated from a tropical climate and if they were male (see Table 4.8).

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Table 4.8 The results of univariable analyses for the variables likely to influence

hookworm infection in dogs from veterinary clinics

Factor Odds ratio (95% CI) P-value

Dogs residing in a tropical 3.5 (1.5, 8.1) 0.002 climate

Dogs not from a tropical 1.0 climate

Male dogs 2.6 (1.0, 7.3) 0.049

Female dogs 1.0

Dogs which had not received 1.0 (0.99, 1.1) 0.067* anthelmintic treatment

Dogs which had received 1.0 anthelmintic treatment

* P- value is not significant but was included for reference

4.3.4.3 Univariable analysis for cats

Only univariable analyses were conducted for the cats as there were too few samples positive for hookworm to enable multivariable analysis. Variables were considered significant if the P- value was ≤ 0.05. Cats from refuges, cats originating from a tropical climatic zone, and those cats which had not received anthelmintics were all considered significant factors via univariable analyses for hookworm infection (Table

4.9).

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Table 4.9. The results of univariable analyses for the variables likely to influence

hookworm infection in cats

Odds ratio (95% CI) P-value

Factor

Cats from refuges 16.4 (2.1, 125) <0.001

Pet cats 1.0

Cats residing in a tropical 8.4 (2.6, 26.9) <0.001 climate

Cats not from a tropical 1.0 climate

Cats which had not received 22.7 (3.0, 166.7) <0.001 anthelmintics treatment

Cats which had received 1.0 anthelmintic treatment

4.4 Discussion

The prevalence of hookworm reported in this study is considerably lower than prevalences reported in previous studies (Table 4.1). The most obvious explanation for this result is the widespread use of anthelmintics in the last 10-15 years, not only in pet dogs and cats but also in animals kept in refuges.

Another significant finding of the present study was the identification of A. ceylanicum in Australia. All of the species of hookworm which may infect Australian dogs and cats are believed to have been introduced in the last 200 years with the arrival of European settlers and their domestic animals (Beveridge, 2002). The dingo was probably introduced 4,000 years ago, but the parasites that accompanied it are unknown (Beveridge, 2002). It was puzzling that A. ceylanicum had not been found previously in Australia given that A. braziliense , A. caninum and A. tubaeforme have all been recovered from Australian dogs and cats in the past (Traub et al ., 2007).

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Furthermore, the absence of A. ceylanicum from northern Australia was intriguing given the endemic distribution of this parasite in neighbouring regions: Malaysia

(Yoshida et al ., 1973; Sheikh et al ., 1985), Borneo (Choo et al ., 2000), Indonesia

(Margono et al ., 1979), Papua New Guinea (Anten and Zuidema, 1964) and the

Solomon Islands (Beveridge, 2002). Finding A. ceylanicum in Australia is of public health significance since it is the only hookworm species capable of infecting dogs and cats which has been shown to produce both experimental and naturally patent infections in humans (Areekul et al ., 1970; Chowdhury and Schad, 1972).

Ancylostoma braziliense was the only species of hookworm not recovered from the present study. Although this species of hookworm is known to occur in Australia it has only rarely been reported in previous studies (Heydon, 1929; Seddon, 1958;

Heydon and Bearup, 1963; Setasuban and Waddell, 1973; Kelly and Ng, 1975;

Taveros, 1990).

Important risk factors for hookworm infection for both dogs and cats included climate, the age of the animal and whether the dog or cat was a pet or had originated from a refuge (Table 4.7, 4.8 and 4.9). A considerably higher prevalence of hookworm infection was found in those animals originating from a tropical climate (Table 4.4).

With the exception of U. stenocephala , warm temperatures and adequate moisture to prevent desiccation provide the most optimal environment for hookworm egg hatching and larval development (Dunsmore and Shaw, 1990). In contrast, U. stenocephala requires lower temperatures for hatching and development (Dunsmore and Shaw,

1990) and this is reflected in the geographical distribution of this parasite (Table 4.5).

Higher parasite prevalences are often noted in younger animals, most likely because of their immature immune systems and also because of transmammary infection

105 routes of A. caninum . As dogs grow older, they become more resistant to hookworms whether or not they experience infection (Bowman et al ., 2003).

Animals originating from refuges are at greater risk of hookworm infection for a multitude of reasons. Many refuge animals have originated from households where they were provided with minimal care, and anthelmintic administration is unlikely.

These animals may also have had a history of roaming and hunting prior to being placed in the refuge and hence could have had exposure not only to contaminated environments but may have also eaten paratenic hosts. Carelessly managed refuges, where faeces are allowed to accumulate, have also been associated with large amounts of infective larvae (Bowman et al ., 2003).

In conclusion, the results of this study demonstrate that it is imperative to have current information regarding the prevalence of parasites and the risk factors associated with infection. This will allow for the more effective implementation of strategic control programmes (Eckert, 1997).

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Chapter 5

Determining the zoonotic significance of Giardia and

Cryptosporidium in Australian dogs and cats

This chapter is a published paper: PALMER, C. S., TRAUB, R. J., ROBERTSON, I. D.,

DEVLIN, G., REES, R. & THOMPSON, R. C. (2008) Determining the zoonotic significance of Giardia and Cryptosporidium in Australian dogs and cats. Veterinary Parasitology, 154 ,

142-7.

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5.1 Introduction

The occurrence of Giardia duodenalis and Cryptosporidium spp. in dogs and cats is of potential significance from both clinical and public health perspectives. We recently reported the results of a national study of intestinal parasites in healthy pet dogs and cats in Australia, where Giardia was found to be the most prevalent parasite in dogs

(9.4%) (Palmer et al ., 2008a). In the same study 0.6% of dogs were found positive for

Cryptosporidium , while the prevalence of Giardia and Cryptosporidium reported in cats was 2.0% and 2.4% respectively.

Although infection with Giardia is common, most dogs and cats remain asymptomatic. Yet infection, regardless of the clinical manifestation, is usually associated with a degree of intestinal pathology which can result in chronic malabsorption. Giardia has been reported to cause villus atrophy, diffuse shortening of microvilli, reduced disaccharidase activity, loss of epithelial barrier function, increased permeability and enterocyte apoptosis (Buret, 2007).

Cryptosporidium infection in dogs and cats does not appear to be common. However, oocyst shedding is more common in younger animals and stress can induce shedding in adult animals, suggesting that chronic, sub-clinical infections could be more common than surveys indicate (Thompson et al ., 2005). Cryptosporidium infection is associated with villus atrophy, villus fusion and inflammation (Koudela and Jiri,

1997) which results in loss of absorptive surface area and impaired nutrient transport.

It is well known that dogs can harbour infections with either zoonotic or host-specific assemblages of Giardia (Caccio et al ., 2005; Thompson et al ., 2008), and this has been demonstrated in a number of recent studies in urban areas of Mexico, Brazil,

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Japan, Italy, Poland and Thailand (Berrilli et al ., 2004; Itagaki et al., 2005; Lalle et al ., 2005a; Eligio-Garcia et al ., 2005; Volotao et al ., 2007; Inpankaew et al ., 2007). In a recent report from Germany it was found that of 60 Giardia positive samples collected from dogs in urban areas, 60% were infected with zoonotic Giardia from assemblage A, 12% with dog-specific assemblages C and D, and the remaining 28% harboured mixed infections (Leonhard et al ., 2007).

Few studies have been undertaken in cats but Vasilopulos et al . (2007) examined 250 cats from Mississippi and Alabama, USA, and of 17 positive for Giardia found six infected with Assemblage A-I and 11 with Assemblage F (the cat genotype).

Recent molecular epidemiological studies of Cryptosporidium in dogs and cats have shown that they seem to be almost exclusively infected with the host-adapted species

C. canis and C. felis respectively (Xiao et al ., 2007). The greatest zoonotic concern regarding these two species of Cryptosporidium is in immunocompromised persons.

In human immunodeficiency virus (HIV) infected people, infections with C. canis and

C. felis have been associated with chronic diarrhoea (Cama et al ., 2007). Chronic diarrhoea in such individuals can lead to a wasting syndrome and eventually death

(Cama et al ., 2007).

The aim of this current study was to determine the zoonotic significance of the

Giardia and Cryptosporidium isolates recovered during a national survey of intestinal parasites in dogs and cats in Australia.

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5.2 Materials and Methods

5.2.1 Samples collected

Single faecal samples were collected from dogs and cats from across Australia, from both urban and rural locations. A total of 1400 canine and 1063 feline faecal samples were collected from 59 veterinary clinics and 26 refuges: refuges (dogs n=590, cats n=

491), and dogs/cats presented to veterinary clinics without gastrointestinal (GI) complaints (dogs n=810, cats n=572). On the day of collection faecal samples were preserved separately in 10% formalin for microscopic screening and 20% dimethyl sulfoxide (DMSO) for molecular screening. There was a time lag of up to 3 months before samples were sent to the laboratory. Once at the laboratory samples were refrigerated at 4°C and the time taken before samples were processed was highly variable, ranging from immediately to 6 months.

Different host factors were recorded for each animal sampled, for these details refer to

Palmer et al . (2008a).

5.2.2 Parasitological procedures

Formalised faecal samples were screened using a standard sedimentation in water technique, a Malachite Green stain for Cryptosporidium oocysts (Elliot et al ., 1999) followed by saturated salt and D-glucose centrifugal flotation and microscopy, as previously described by Henriksen and Christensen, (1992).

5.2.3 Molecular methods

Genotyping of Giardia was initially performed at the 18S rDNA locus for a subset of

88 of the 131 microscopy positive dog samples. This subset consisted of 43 of the 44

110 positive samples from veterinary clinics and 45 randomly selected samples of the 87 positive samples from refuges. A decision was made not to amplify all of the microscopy positive samples due to limited time and expense, and because we were finding very consistent results. For confirmation of the assemblages determined using this locus, a selection of these dog samples were also amplified at the beta giardin (β- giardin) locus.

Because the products produced by the primers used for the 18S rDNA are identical for both assemblage A and assemblage F, all of the Giardia positive cat samples were only amplified at the β-giardin locus.

PCR amplification of the actin gene locus was performed for all of the samples found positive for Cryptosporidium on microscopy.

5.2.3.1 DNA extraction

Two hundred micrograms of faeces was suspended in 1.4 ml ATL tissue lysis buffer

(Qiagen, Hilden, Germany). This suspension was then subjected to five cycles of freeze-thawing at liquid nitrogen temperatures followed by boiling for 10 minutes.

DNA was then isolated from the supernatant using the QIamp DNA Mini Stool Kit

(Qiagen, Hilden, Germany) according to manufacturer’s instructions. Final elutions of

DNA were made in 50 µl of elution buffer instead of 200 µl as recommended by the manufacturer.

5.2.3.2 PCR amplification and sequencing of the Giardia 18SrDNA

A nested PCR was used for amplification at the 18SrDNA locus. For the primary reaction, primers RH11 and RH4 (Hopkins et al ., 1997) were used and for the

111 secondary reaction primers GiarF and GiarR (Read et al ., 2002) were used. The amplification conditions for both sets of primers were the same as those described by

(Hopkins et al ., 1997).

Products were isolated from agarose gel using a DNA purification kit (Mo Bio,

UltraClean GelSpin, U.S.A.) as per manufacturer’s instructions, except for a reduced elution volume. Sequencing reactions were performed without DMSO, using the Big

Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions. PCR products were sequenced in the reverse direction only (GiarR). Reactions were electrophoresed on an ABI 3730 48 capillary machine.

Sequences were analysed using Finch TV Version 1.3.1 (Geospiza Inc.) and isolates were grouped into their genetic Assemblages based on their polymorphisms within the

130 base pair sequence (Hopkins et al ., 1997, Monis et al ., 1999).

5.2.3.3 PCR amplification and sequencing of the Giardia β-giardin gene

The amplification of the β-giardin gene was performed using a nested PCR protocol.

In the primary PCR reaction a fragment was amplified using the forward primer G7 and the reverse primer G759, as previously described (Caccio et al ., 2002). In the sequential nested PCR reaction, the primers described by Lalle et al ., (2005b) were used. Amplification conditions were modified for both the primary and secondary reactions. In both reactions 1µl of extracted DNA was added to 1.5mM MgCl2,

10pmol of each primer, 200µM of each dNTP, 0.5 units of Tth Plus DNA polymerase

(Biotech International, Perth, Australia), 1x reaction buffer (67mM Tris-HCL,

16.6mM (NH4)2SO4, 0.45% Triton X-100, 0.2mg/ml gelatin) and H 2O to 25µl total.

Reactions were denatured at 94°C for 2 min followed by 40 cycles in the primary and

35 cycles in the secondary, each consisting of 30 sec at 94°C, 30 sec of annealing

112

(65°C for the primary and 55°C for the nested) and 60°C at 72°C, followed by a final extension of 7 min at 72°C.

Products were isolated and sequenced and analysed as per the 18SrDNA locus above.

Sequencing was performed with both the forward and reverse primers from the nested

PCR. Sequences were aligned with each other as well as previously published sequences for G. duodenalis isolates using Clustal W (Thompson et al ., 1994).

5.2.3.4 PCR amplification and sequencing of the Crytosporidium actin gene

PCR amplification of the actin gene locus was performed using a nested PCR as previously described (Ng et al ., 2006). Sequencing was performed with both the forward and reverse primers from the nested PCR. Products were isolated, sequenced and aligned as described above.

5.2.4 Statistical analysis

The methods used to determine any association between parasitism and host factors are outlined in Palmer et al . (2008a).

5.3 Results

5.3.1 Microscopy

For the majority of microscopy positive Giardia and Cryptosporidium samples the quantity of cysts/oocysts was low. Frequently there was less than one cyst/oocyst observed per 40X field of view.

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5.3.2 Risk factor analysis

Multivariable analysis revealed that dogs from refuges and dogs which were less than one year of age were at greatest risk of Giardia infection. Statistical analysis was not performed on the dogs positive for Cryptosporidium and the cats positive for either

Giardia or Cryptosporidium , as there were too few positives.

5.3.3 Molecular characterization of Giardia and Cryptosporidium isolates

It should be mentioned that genotyping of the microscopy positive Giardia samples was initially attempted at the triose phosphate isomerase (tpi) gene and the glutamate dehydrogenase (gdh) gene, using primers previously described by Sulaiman et al .

(2003) and Read et al . (2004) respectively. Unfortunately, all attempts at these two loci were unsuccessful. Amplification of the tpi gene resulted in non-specific banding patterns, while the ghd primers were unable to amplify any of the samples apart from the positive control (axenic culture of the G. duodenalis Portland 1 strain).

Of the 88 Giardia positive dog isolates sequenced at the 18SrDNA locus, 46.6% (41) were Assemblage C, 50% (44) were Assemblage D, 2.2% (2) were mixed genotype C and D, and 1.1% (1) was Assemblage A. A subset of the isolates (n = 12) were also genotyped at the β-giardin locus and confirmed the 18S results.

Of the 21 Giardia positive cat samples 8 were successfully sequenced at the β-giardin locus, seven of which were Assemblage F and one was Assemblage D.

Phylogenetic analysis for subgrouping genotypes was determined not to be necessary as no Giardia Assemblages A or B were recovered using the β-giardin gene. Although one dog isolate was found to be Assemblage A using the 18SrDNA locus we were

114 unable to amplify this sample at the β-giardin gene and the 18S fragment is too small to perform phylogenetic analysis on.

Four of the 8 Cryptosporidium positive dog specimens where successfully sequenced and were found to be C. canis . Eighteen of the 26 positive cat isolates were sequenced and all were identified as C. felis .

5.4 Discussion

Risk factor analysis revealed that dogs from refuges and dogs which were less than one year of age were at the greatest risk of Giardia infection. Refuge dogs are at more risk of infection due to the direct contact they have with other dogs and their excrement, environmental contamination and the immunosuppressive effects of stress.

Younger animals are at risk of infection due to their immature immune systems.

It was disappointing that neither of the primer sets for the Giardia tpi and gdh genes were able to successfully amplify any of the isolates. The sensitivity of the β-giardin locus was also very poor as evident by the few cat isolates we were able to amplify. In contrast, we had great success with the 18SrDNA locus and according to Wielinga and

Thompson (2007) the 18SrDNA locus is well-suited for routine genotyping from environmental samples due to its high copy number; whereas the other structural and metabolic genes are estimated to be single or low copy numbers. Environmental samples are typically low in target DNA and contain many PCR inhibitory factors

(Wielinga and Thompson, 2007). Castro-Hermida et al . (2007) reported that when the quantity of cysts was low (less than one cyst per field of view), PCR was unsuccessful at the gdh and β-giardin locuses which fits with our current findings.

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Although it was expected that animals from refuges would be predominantly infected with Giardia host-specific genotypes, the low prevalence of zoonotic genotypes was unexpected in animals from households. Thompson et al . (1999) found that in the domestic, urban environment of Perth Western Australia, genotypes from Assemblage

A and C were both equally common in the dogs sampled. It has been suggested that in environments where the infection pressure is low, such as in domestic households in urban settings, dogs are just as likely to harbour genotypes from Assemblage A as they are their own host specific genotype (Assemblage C and D) (Thompson and

Monis, 2004). In contrast, in Aboriginal Communities in Australia, where Giardia infection is highly endemic for both the human and dog populations it was found that the dog genotype predominated in infected dogs (Hopkins et al ., 1997). It is most probable that the dogs in these communities would be exposed to both the dog specific as well as the zoonotic genotypes of Giardia , however the dog specific genotypes, which are probably ‘better’ host adapted, are more likely to predominate through competitive exclusion (Thompson, 2000). Traub et al . (2004a) reported that all of the dogs infected with Giardia in a remote tea-growing community in Assam, India, were infected with Giardia from Assemblage A. The difference between Assam and the findings of Hopkins et al . (1997) in the Aboriginal Communities may reflect a closer association between individual dogs and their owners in Assam, and the frequency with which dogs are able to eat human faeces in these communities, as well as the territorial nature of the dogs with little dog to dog contact (Traub et al ., 2004a). A possible explanation for the findings in the household animals in the current study, which was in contrast to the findings of Thompson et al . (1999), is that the dogs and cats in the current study had not been exposed to the zoonotic genotypes because the human population had a low prevalence of Giardia infection. Surveys that simultaneously target both humans and their companion animals with regards to risk

116 factors for the prevalence of zoonotic versus dog-specific genotypes of Giardia will shed further light on this matter.

Unexpectedly, one of the Giardia isolates from a cat was identified as Assemblage D, an Assemblage usually regarded as dog specific. In the literature there is only one other reference to such an occurrence; McGlade et al . (2003b) reported finding

Giardia Assemblage D in 13 of 14 cat isolates sequenced. We were unable to ascertain whether the cysts present in the faeces in the cat in our study were the result of mechanical transmission or a patent infection.

Future research into the possibility of correlating Giardia Assemblage type with differences in pathogenicity is needed. As a starting point samples should be taken from dogs and cats with giardiasis to see if they are mostly infected with a particular

Giardia genotype.

The finding of C. canis and C. felis in dogs and cats respectively was not surprising and supports the limited number of studies conducted previously (Morgan et al ., 1998;

Abe et al ., 2002a; Fayer et al ., 2006; Xiao et al ., 2007). It would thus appear that globally, pet dogs and cats do not represent a public health risk with respect to the transmission of Cryptosporidium to humans unless they are immunocompromised.

In conclusion, although Giardia is the most prevalent parasite in dogs in Australia

(Palmer et al ., 2008a) the zoonotic risk seems to be low. However, the option of genotyping Giardia positive specimens is not readily available to veterinarians in practice; consequently all positives should be assumed zoonotic. Therefore, treatment

117 is necessary regardless of whether the animal is asymptomatic given the zoonotic potential of this parasite, and because they are a source of infection for other animals.

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Chapter 6

Intestinal parasites of dogs and cats in Australia; the

veterinarian’s perspective and pet owner awareness

This chapter has been submitted to the Veterinary Journal as a short communication:

PALMER, C. S., ROBERTSON, I. D., TRAUB, R. J., REES, R. & THOMPSON, R. C.

(2008) Intestinal parasites of dogs and cats in Australia; the veterinarian’s perspective and pet owner awareness.

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6.1 Introduction

Veterinarians have an obligation to provide pet owners with up to date information about parasites which could infect their pets and which are potentially zoonotic.

Although veterinarians are an effective source of information it has been identified that many are providing incorrect, minimal or out of date information (Kornblatt and

Schantz, 1980; Harvey et al ., 1991). Schantz (1994) speculated that veterinarians were reluctant to advise clients about parasites because they were often not confident with the subject matter and do not want to alarm their clients, particularly if this might lead them to give up their pets. More recently, particular blame has been placed on an ever- increasing choice of broad-spectrum anti-parasitic drugs, which has obviated the need for veterinarians to discuss parasites with their clients (Zajec, 2000).

The Centres for Disease Control and Prevention (CDC website, 16/12/07) recommends treatment of puppies and kittens at 2, 4, 6 and 8 and 3, 5, 7, and 9 weeks of age respectively to prevent establishment of Toxocara canis , T. cati and

Ancylostoma caninum infection. Nursing bitches and queens should be prophylactically treated concurrently with their offspring every 2 weeks in order to eliminate reactivated larvae (Misra, 1972) and horizontal transmission from shedding neonates (Lloyd et al ., 1983).

The Companion Animal Parasite Council (CAPC website, 5/8/08) recommends an integrated approach to parasite control in adult animals, preferably by including a year-round treatment with a broad-spectrum heartworm prophylaxis/ intestinal parasite combination product that helps reduce the risk of parasitism in pets and zoonotic parasite transmission.

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6.2 Materials and Methods

Between March 2004 and September 2005 a national study of intestinal parasites in dogs and cats in Australia was undertaken (Palmer et al ., 2007; Palmer et al ., 2008a;

Palmer et al ., 2008b) . In addition, questionnaires were administered to veterinarians and pet owners. In total, 59 veterinary clinics, 676 dog owners and 420 cat owners participated in the study. Of the 59 veterinary clinics, 46 (78%) completed the questionnaire. Our objectives were to evaluate the current deworming protocols used by veterinarians, their perception of GI parasites, and the degree of client education with particular reference to the level of zoonotic awareness by pet owners.

On a scale of 1 to 5 with 1 being ‘not a problem’ and 5 being ‘a significant problem’ veterinarians were asked to rank how much of a problem they considered specific parasites to be in dogs and cats in their practice area. In order to interpret these results, rankings of 1 and 2 were categorised as ‘not a problem’, 3 as ‘a slight problem’ and 4 and 5 were classed as ‘a significant problem’ (Tables 6.1 and 6.4). Similarly, on a scale of 1 to 5 with 1 being ‘not concerned’ and 5 being ‘very much concerned’ vets were asked to rank how concerned they were about the zoonotic hazard of particular parasites, these rankings were then categorised in a similar fashion as above (Tables

6.2 and 6.5). In addition, the questionnaire collected data on recommended deworming protocols and level and means of client education.

The pet owner questionnaire collected data on owner awareness of zoonoses and current use of anthelmintics in their pets. Both the veterinarian and pet owner questionnaires were initially pre-trialled at three selected veterinary practices in an effort to identify any possible problems with the questions included.

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Data were analyzed using the frequencies function in SPSS (SPSS for Windows,

Version 14.0).

6.3 Results

Twenty of the 46 vets considered Dipylidium caninum to be a significant problem in their practice area (Table 6.1) yet the national prevalence was 0.1% (Palmer et al .,

2008a) and it was not recovered from any dogs originating from vet clinics. Although

11 veterinarians considered Toxocara canis to be a significant problem in their practice area and 23 veterinarians were very concerned with it as a zoonotic hazard, the national prevalence was only 1.2% (Palmer et al ., 2008a). Giardia spp. and hookworm species were found to be the most prevalent parasites in dogs nationally

(Palmer et al ., 2008a). Unlike hookworm, the prevalence of Giardia was not shown to significantly vary between climatic zones (Table 6.3) and has previously been reported as a ubiquitous parasite (Thompson et al ., 2008). Nine vets considered

Giardia to be a significant problem in dogs in their practice area, yet the majority of dogs from these practices were not positive for Giardia on faecal examination. In contrast to Giardia , hookworm was found to be more prevalent in locations with a tropical climate (Table 6.3). Of the 8 vets who considered hookworm to be a significant problem in their practice area, 5 originated from Brisbane, 1 from Darwin,

1 from Broome and 1 from Sydney. Of the 12 vets who were very concerned with the zoonotic hazard posed by hookworm, 5 also thought it was a significant problem in dogs in their practice area.

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Table 6.1 How much of a problem veterinarians consider the following parasites

in dogs in their practice area

number of veterinarians

Parasite Not a A slight A significant Don’t know

problem problem problem

Toxocara canis 18 15 11 2

Hookworm 26 10 8 1

Echinococcus 36 4 5 0 granulosus

Dipylidium 15 10 20 1 caninum

Taenia /Spirometra 28 8 3 7

Trichuris vulpis 31 7 5 3

Giardia spp. 22 10 9 5

Cryptosporidium 22 5 0 19 spp.

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Table 6.2 Veterinarians concern for the zoonotic hazard of the following

parasites in dogs in their practice area

number of veterinarians

Parasite Not a concern Slightly Very much Don’t know

concerned concerned

Toxocara canis 14 9 23 0

Hookworm 27 7 12 0

Echinococcus 24 7 14 0 granulosus

Dipylidium 36 6 4 0 caninum

Taenia /Spirometra 32 7 3 4

Trichuris vulpis 39 4 1 1

Giardia spp. 16 12 15 3

Cryptosporidium 17 9 5 15 spp.

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Table 6.3 Prevalence of hookworm and Giardia in dogs from veterinary clinics

and refuges in major cities and regional areas in Australia

City/Town Climate Prevalence of Prevalence of Number of

Giardia % (95% hookworm % faecal samples

CI) (95% CI) examined

Adelaide Temperate 4.2 (0-8.8) 0 72

Brisbane Tropical 4.5 (1.2-7.8) 11.8 (6.7-16.9) 154

Broome Tropical 3.6 (0-8.5) 25.0 (13.7-36.3) 56

Cairns Tropical 0 24.4 (11.3-37.6) 41

Canberra Temperate 8.2 (2.4-14.0) 2.4 (0-5.7) 85

Darwin Tropical 13.6 (3.5-23.7) 29.5 (16.0-43.0) 44

Gold Coast Tropical 6.7 (0-19.4) 13.3 (0-30.5) 15

Hobart Temperate 8.0 (1.9-14.1) 2.7 (0-6.4) 75

Melbourne Temperate 8.0 (3.7-12.3) 2.0 (0-4.2) 150

Perth Temperate 10.7 (5.0-16.4) 1.8 (0-4.3) 112

Sunshine Coast Tropical 14.3 (4.5-24.1) 10.2 (1.7-18.7) 49

Sydney Temperate 15.1 (8.9-21.3) 1.6 (0-3.8) 126

Seventeen vets considered D. caninum to be a significant problem in cats in their practice area (Table 6.4), yet this parasite was not recovered from any of the cats originating from veterinary clinics and in only 0.4% of refuge cats in the national study (Palmer et al ., 2008a). Vets were most concerned with the zoonotic hazard of

Toxoplasma gondii infection in cats (Table 6.5).

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Table 6.4 How much of a problem veterinarians consider the following parasites

in cats in their practice area

number of veterinarians

Parasite Not a A slight A significant Don’t know

problem problem problem

Toxocara cati 20 13 9 1

Hookworm 32 6 4 1

Dipylidium 16 10 17 0 caninum

Taenia /Spirometra 24 10 5 4

Giardia spp. 24 11 0 9

Cryptosporidium 23 4 0 16 spp.

Toxoplasma 15 12 4 13

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Table 6.5 Veterinarians concern for the zoonotic hazard of the following

parasites in cats in their practice area

number of veterinarians

Parasite Not a concern Slightly Very much Don’t know

concerned concerned

Toxocara cati 19 11 13 0

Hookworm 32 5 5 0

Dipylidium 35 6 2 0 caninum

Taenia /Spirometra 33 6 3 1

Giardia spp. 21 10 6 5

Cryptosporidium 20 5 5 13 spp.

Toxoplasma 14 6 19 4 gondii

The deworming recommendations made by the 46 participating vets are summarised in Table 6.6. Seventeen of the vets recommended a product for the treatment or protection of hookworm infection in dogs and cats which relied solely on the activity of pyrantel embonate, a drug that has recently shown to display poor efficacy against

Brisbane isolates of Ancylostoma caninum (Kopp et al ., 2007).

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Table 6.6 Veterinarians recommendations (n= 46) concerning the prophylactic

administration of anthelmintics for the control of GI parasites

Number of veterinarians

Veterinarians Puppies Pregnant or Adult Kittens Nursing or Adult recommendations nursing dogs pregnant cats

bitches queens

Recommend specific 46 40 43 43 33 41 prophylactic program against intestinal parasites

Recommend 37 deworming from 2 weeks of age until 12 weeks of age at 2 weekly intervals

Recommend 37 deworming prior to 6 weeks of age until at least 10 weeks of age at 2 weekly intervals

Recommend 32 27 administering anthelmintic to pregnant dog or cat

Recommend 9 5 administering anthelmintic to lactating mother at the same time as puppies or kittens

Recommend monthly 5 7 prophylactic treatment with a product effective against GI parasites and heartworm

Recommend three- 39 34 monthly prophylactic administration of anthelmintic

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Recommend six- 12 12 monthly prophylactic administration of anthelmintic

Recommend examining 1 1 1 1 1 1 a faecal sample prior to anthelmintic administration

Few vets routinely discussed the zoonotic potential of all parasites with all clients

(Table 6.7).

Table 6.7 Frequency with which the veterinarians surveyed (n=46) discussed the

potential zoonotic hazards with their clients of the following parasites

Number of veterinarians

Roundworm Hookworm Tapeworms Protozoa

Only when asked 5 8 7 11

Whenever worms are 23 19 20 19 diagnosed in clients pets

Routinely with new clients 12 8 10 8

Routinely with clients with 31 22 23 13 puppies or kittens

Routinely with clients known 29 22 19 11 to have children

Routinely with all clients 6 2 4 3

The most common source of information for veterinarians on anthelmintics and

dosing regimes was from visiting pharmaceutical drug company representatives,

followed by advertising material produced by drug companies.

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Of the dog and cat owners who participated in this study 71.3% and 75.5%, respectively, were aware of zoonoses (Table 6.8). The most common source of information for pet owners pertaining to zoonoses was the veterinarian (Table 6.9). Of the 58.8% of owners who stated that veterinarians had educated them about zoonoses,

24.8% had solely been educated by veterinarians (Table 6.9).

Table 6.8 Dog owner (n = 676) and cat owner (n = 420) awareness of zoonoses

General zoonotic awareness Dog owner Cat owner

% %

Aware of zoonoses 71.3 75.5

Aware zoonoses can be contracted from picking up 80.2 93.1 pet faeces

Aware zoonoses can be contracted from contact 56.7 62.9 with soil, lawn or plants with which pets have also had contact

Awareness of specific parasites

Toxocara 71.3 70.7

Hookworm 55.6 47.8

Dipylidium caninum 24.9 33.8

Echinococcus granulosus 74.9

Trichuris vulpis 16.7

Giardia spp. 50.2 48.4

Cryptosporidium spp. 25.8 24.5

Toxoplasma gondii 68.2

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Table 6.9 Source of information for pet owners regarding zoonoses

Total Medical Vet School/ Breeding Pet Friends/ Health TV Radio

Doctor Technical college/ Society Shop family Department

University

Medical Doctor 13.6 1.2 11.9 2.1 1.6 2.0 6.2 2.0 4.0 1.4

Vet 58.8 11.9 24.8 10.3 3.0 4.7 13.7 4.0 10.9 3.2

School/ 19.1 2.1 10.3 4.2 1.1 1.4 3.7 1.3 2.2 0.8 Technical college/ University

Breeding 4.6 1.6 3.0 1.1 0.7 0.7 1.4 0.7 1.5 0.6 society

Pet shop 5.2 2.0 4.7 1.4 0.7 0.4 2.7 0.7 2.1 0.8

Friends/ 26.7 6.2 13.7 3.7 1.4 2.7 9.6 1.4 5.4 1.7 Family

Health department 5.5 2.0 4.0 1.3 0.7 0.7 1.4 0.1 2.2 0.7

TV 15.1 4.0 10.9 2.3 1.6 2.1 5.4 2.2 2.4 3.8

Radio 4.0 1.4 3.3 0.8 0.6 0.8 1.7 0.7 3.8 0.1

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The anthelmintic history for all dogs and cats which participated in the study is summarised in Table 6.10 .

Table 6.10 Anthelmintic history for dogs (n = 810) and cats (n = 572) from pet

owner questionnaire

Anthelmintic history Dogs Cats

% %

Received anthelmintic in 81.2 86.7 the previous 12 months

Anthelmintic administered 34.0 31.6 which solely relies on pyrantel for activity against nematodes

Anthelmintic administered 54.0 39.1 at three-monthly intervals

Anthelmintic administered 9.3 15.9 monthly which is effective against both intestinal worms and heart worm

6.4 Discussion

Few of the vets surveyed routinely discussed the zoonotic potential of all dog and cat

GI parasites with all clients. This was reflected by not all pet owners being aware of zoonoses. Previous research has shown that physicians rely on veterinarians to educate the public about the risk and methods of preventing zoonotic disease (Grant and

Olsen, 1999). Australian veterinarians have an important role in the community as educators however they seem to fall short when it comes to promoting zoonotic awareness.

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Most of the veterinarians adhered to advocating the regular prophylactic administration of anthelmintics throughout a pet’s life and most recommended products with broad spectrum activity. Perhaps as a result, veterinarians no longer see the need to, or are more complacent, about discussing the zoonotic risk associated with parasites.

Apart from hookworm most of the surveyed veterinarians were largely unaware of the parasite situation in their area which is not surprising given that most of them do not examine faecal samples prior to anthelmintic administration and there is no current published data on regional prevalences to refer to. It could be speculated that veterinarians do not perceive the importance of being aware of the parasite situation in their local area due to their reliance on anthelmintics. This approach is reinforced by the low prevalence of GI parasites in Australia, a situation which has most likely arisen as a result of the widespread prophylactic administration of anthelmintics, and consequently veterinarians see few clinical cases of GI parasitism.

A significant number of the participating veterinarians recommended a product which solely relied on the action of pyrantel embonate for the treatment/protection of dogs and cats from hookworm infection, and in conjunction approximately 30% of pet owners had used such a product. A high level of pyrantel resistance in A. caninum was recently documented in dogs in Brisbane, Australia (Kopp et al ., 2007). A lack of awareness of the parasite situation in an area may result in failure to detect resistance when it occurs. Furthermore, it has been shown that some generic allwormers contain sub-optimal levels of pyrantel which could also hasten the development of drug resistance (Hopkins et al ., 1998).

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Veterinarians are losing the opportunity to be educators and there is over reliance on anthelmintics to prevent and control parasites and their zoonotic risk. Given the low prevalence of parasites in Australia, Australian veterinarians should consider conducting periodic faecal exams in pets in order to determine whether anthelmintic treatment is warranted. This will allow de-worming practices to be more sensitive to regional prevalence and lead to a reduction of selection pressure for anthelmintic resistance. This approach inherently is associated with a degree of risk and it will be imperative that veterinarians concurrently educate clients on good hygiene practices including the prompt removal of faeces and the confinement of pets to reduce behaviours such as hunting and scavenging as a means of reducing the risk of pet parasitism and zoonoses.

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Chapter 7

General Discussion

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7.1 Gastrointestinal parasites in pet dogs and cats in Australia

From the current study it was clear that most Australian veterinarians were not aware of the prevalence of parasites in Australia. It is hoped that this research has now shed more light on the matter and revealed that, for the most part, the prevalence of intestinal parasites in pet dogs and cats is low. However, despite this overall finding there remains areas were the prevalence of parasites is high, for example, hookworm infection was considerably higher in the tropical regions of the country, particularly in refuge dogs. Echinococcus granulosus remains a concern in focal hyperendemic areas, however it was not within the limits of this study to determine the prevalence given its localised distribution, particularly in farm working dogs which are unlikely to be taken to veterinary clinics (Jenkins et al ., 2006). The unique life cycle of this parasite, its public health significance, and its localised distribution warrant further targeted surveillance.

Giardia was determined to be the most prevalent parasite in dogs and the prevalence recorded (9.3%) is likely to be a gross underestimation of the true prevalence due to the intermittent nature in which cysts are shed (Sherding, 1983). It is concerning that

22 veterinarians (Table 6.1), of the 46 surveyed, did not believe the dogs in their practice area were at risk of infection with Giardia. Only three veterinarians (Table

6.7) discussed the zoonotic potential of protozoa with all their clients. The majority of the veterinarians surveyed recommended the control of GI parasites in dogs and cats through the routine administration of an ‘allwormer’ (broad spectrum anthelmintic).

However, ‘allwormers’ do not prevent infection with Giardia or other protozoal infections and as a consequence of their use, client education, an effective means of parasite prevention, is often overlooked.

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It is important to acknowledge that the low prevalence of parasites recorded is most likely a result of the high rate of prophylactic administration of anthelmintics to pets.

It could be argued that because such an approach has been so effective it should be continued, and if it was not, it would result in a rise in the prevalence of intestinal parasites. I would argue that it is not good veterinary science to prescribe anthelmintics to animals which are possibly parasite-free or which have a low risk of acquiring infection, and that Australia is in a favourable position to effectively rely on the power of client education as a means of preventing infection. To add to the argument, when parasites are a considerable problem, inappropriate use of anthelmintics due to a lack of awareness of parasite prevalence, could facilitate drug resistance. A standardised approach to control through the regular prophylactic administration of anti-parasitic drugs prevents the veterinarian from determining the parasite situation in their area and does not foster pet owner education. Overreliance on anthelmintics can result in veterinarians becoming complacent about parasites and subsequently they may be overlooked in differential diagnoses.

7.1.1 The need for more emphasis on education

My study revealed that the veterinarian is the most common source of information pertaining to zoonoses for the pet owner; however, most veterinarians did not routinely discuss parasites with all pet owners. The study also revealed that veterinarians were largely unaware of the parasite situation in their local area and that drug companies were their main source of information on parasites. As outlined in the previous chapter it has been speculated that a lack of confidence in the subject matter, a desire not to alarm clients (Schantz, 1994) and an overreliance on broad spectrum anthelmintics (Zajac et al ., 2000) have all contributed to the reluctance of veterinarians to discuss parasites and parasite control with pet owners.

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Future educational campaigns need to be targeted at veterinary students who hopefully will then disseminate the information to future clients. It has been stated that as a consequence of parasitology being taught in the initial years of a veterinary degree, and not re-emphasised in the later years, its importance becomes redundant (Zajac et al ., 2000). For many veterinarians in small animal practice, management of parasites is merely through the use of anti-parasitic drugs (Zajac et al ., 2000). Through re- emphasising parasites and their lifecycles (clinical and zoonotic importance) in the final years of a veterinary curriculum, veterinarians could be instructed that the main means of parasite control and prevention should be through good husbandry and proper animal management, including, reducing contact between pets and intermediate hosts. Graduating veterinarians are not only a source of information for pet owners but also a source of information for their colleagues; thereby targeting the veterinary student in an educational campaign maximises the impact that could potentially be made.

7.2 Acknowledging the limitations of the current study

7.2.1 Dogs and cats sampled

Although the collaboration with BayerHealth, Australia, was advantageous and allowed for the collection of samples throughout Australia, there were also several limitations to using this approach. Namely, sample collection was limited to collecting from veterinary clinics and refuges and samples were not collected from pet shops or breeding facilities. Puppies and kittens in pet shops and animals in breeding colonies have been shown to be at risk of parasitic infection (Bugg et al ., 1999; McGlade et al .,

2003a). Importantly, puppies and kittens purchased from these facilities are a potential source of zoonoses and are most susceptible to the deleterious effects of parasites.

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A potential bias for the samples collected at the participating veterinary clinics is that the pet owners who volunteered to participate in the study may have done so because they are particularly diligent pet owners, whose animals are extremely well looked after. On the contrary veterinarians may have encouraged certain pet owners to participate as they believed their pet to be potentially parasitised.

The refuges who participated in the study did so because they had a relationship with

Bayer, who provides pro-bono anti-parasitic drugs. This is of potential bias as the animals in these refuges may not be representative of animals in other refuges.

Although many refuges which re-home animals provide anti-parasitic drugs, potential differences may arise as a result of using inferior products and inappropriate worming regimes, most likely as a consequence of the associated high costs with using a more rigorous regime.

7.2.2 Sampling

The prevalence of parasites reported in this study was based on the collection of only one faecal sample from each individual and hence it is likely to be an underestimate of the prevalence, given that many parasites intermittently shed eggs/cysts, and some infections may be prepatent. Unfortunately it was not feasible to collect more than one faecal sample over consecutive days given the magnitude of the study.

The effect of climate on the distribution and intensity of parasites is well documented

(Gemmell, 1958; Dunsmore et al ., 1984; Oliveira-Sequeira et al ., 2002). Australia spans many different climatic zones and the results revealed that the prevalence of hookworm varied significantly between tropical and temperate climates. In order to most accurately determine the sample prevalence within 5% with a 95% confidence interval (with an assumed prevalence of 50%) a sample size of 384 was required for 139 both species sampled. Although it was possible to determine the prevalence of parasites in a temperate climate versus a tropical climate for dogs, beyond this further breakdown of regional prevalences was not possible for either dogs or cats because of insufficient samples. Ideally it would have been beneficial to determine the prevalence of parasites in the major cities and rural centres; however this would have required substantially more sampling from each location which was not feasible as part of a doctoral program.

7.2.3 Methodology

The centrifugal flotation technique used in this study, while effective for recovering many helminth eggs and protozoan cysts, is likely to have resulted in an underestimation of the prevalence of parasites. Flotation techniques are considered unreliable for the detection of light infections, nematode larvae, trematodes and the proglottids of cestodes (Bowman et al ., 2003). However, it must be noted that in this study the larvae of A. abstrusus and C. aerophila were detected using saturated salt and D-glucose centrifugal flotation.

The gold standard for detection of parasites is the examination of animals by necropsy. Necropsy not only allows for the detection of prepatent and patent infections but also allows for the detection of any pathology associated with infection.

Performing necropsies are very time consuming and only a limited number of animals can be sampled, particularly as concerns over animal welfare by the community leads to active discouragement of the use of animals for research purposes.

7.2.4 Limitations in perspective

Thirty five urban and rural locations from throughout Australia were included in the study. Representation of such diverse geographical locations is almost unprecedented 140 and has resulted in collection of significant national data. Similar epidemiological studies conducted internationally are usually limited to a particular geographical area

(Dubna et al ., 2007; Inpankaew et al ., 2007; Martinez-Carrasco et al ., 2007; Miro et al ., 2007; Morishima et al ., 2007) or a particular demographic group, for example shelter/refuge dogs (Blagburn et al ., 1996). The estimates of prevalence are also often based on small sample sizes and consequently the accuracy of such studies is often lower than studies with a larger sample size (Jovani and Tella, 2006), such as was the case in my study.

A limitation of many of the previous studies conducted in Australia is that they only examined refuge/shelter animals (Setasuban and Waddell, 1973; Kelly and Ng, 1975;

Pavlov and Howell, 1977; Blake and Overend, 1982; Wilson-Hanson and Prescott,

1982; Shaw et al ., 1983; Collins et al ., 1987). Prevalence results obtained from animals only from refuges/shelters gives an unrealistic picture of the prevalence of parasites in the general pet population, as animals from these locations are at greatest risk of parasitic infection. The question I wanted to answer in the current study was

‘what is the prevalence in the general pet population of Australia?’ as these are the animals for which humans are at greatest exposure to and anti-parasitic drugs are marketed at. In consideration that not all pets are taken to veterinary clinics, and in order to represent both well cared for pets as well as potentially less cared for animals, samples were collected from not only veterinary clinics but also refuges.

7.3 The power and conversely the limitations of using molecular tools

The ability to genetically characterise parasitic stages directly from faeces was a major advantage of the current study. Molecular tools were applied in combination with classical parasitological methods to detect and genetically characterise parasites such as hookworm, Giardia duodenalis and Cryptosporidium spp.. Molecular tools 141 alleviated the need for tedious morphological identification of adult hookworms following anthelmintic purging or at necropsy and also the need for laborious laboratory amplification of Giardia using in vitro or in vivo cultivation techniques.

The use of molecular tools allowed for the identification of A. ceylanicum for the first time in Australia and provided insight into the zoonotic potential of Giardia and

Cryptosporidium positive samples.

A number of studies have used molecular screening of faeces to determine the prevalence of parasites such as Giardia and Cryptosporidium (McGlade et al ., 2003b;

Giangaspero et al ., 2006). The very high sensitivity of most PCR-based procedures can present problems of interpretation (Thompson, 2004) and as a consequence it was decided molecular screening of samples to determine prevalence would not be undertaken. For example, McGlade et al. (2003b) detected a prevalence of 5% of

Giardia in domestic cats using zinc sulphate flotation and microscopy yet when PCR was applied to the same samples 80% were found to be positive. Thompson (2004) suggested that such results as those obtained by McGlade et al . (2003b) could not be accounted for by the intermittent nature of cyst shedding by Giardia and raised questions concerning both the clinical and epidemiological significance of such presumably low-level infections with Giardia .

There is definitely a place for molecular tools in aiding diagnosis via traditional microscopic techniques. However, PCR alone is not practical for routine parasitology where the ability to detect multiple parasites is required. Furthermore, PCR is costly and requires specialised equipment, which tends to limit its application to research or specialised diagnostic laboratories (O'Handley and Olson, 2006).

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7.4 Future research

7.4.1 Giardia

A considerable amount of research has been undertaken on Giardia , yet despite this the mechanism of pathogenicity and the host response to infection remain uncertain

(Roxstrom-Lindquist et al ., 2006). A puzzling feature of infection with Giardia is that many infected animals remain asymptomatic while others present with clinical signs such as diarrhoea. Many factors have been proposed to account for the disease variability such as strain genotype (Aggarwal and Nash, 1987; Nash et al ., 1987), state of the immune system, host age and nutritional status, infectious dose (Faubert, 2000) and, possibly co-infections (Singer and Nash, 2000); these areas should remain an important focus for future research.

Although, it is established that certain genotypes of Giardia are zoonotic (Monis and

Thompson, 2003), in the present study the overwhelming majority of genotypes amplified were dog or cat specific. For household dogs and cats it was postulated that the lack of zoonotic genotypes recovered was as a consequence of there being little infection in the human population. Previous epidemiological surveys, which have looked at humans and dogs living in the same area, have demonstrated identical genotypes of G. duodenalis in dogs and their owners (Traub et al ., 2004a; Inpankaew et al ., 2007). Unfortunately, subgenotyping tools that are capable of discriminating origins or sources of transmission for Giardia do not yet exist (Traub, 2008), and the question of ‘who infected who’ often remains unresolved. Data on the frequency of zoonotic transmission and the exact role of animals as reservoirs are lacking (Traub,

2008). Future epidemiological surveys involving both humans and their pets will continue to shed more light on this matter.

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7.4.2 Echinococcus granulosus

Tasmania was declared provisionally free of E. granulosus in 1996; 30 years after the

Tasmanian hydatid control campaign began (Beard et al ., 2001). During the campaign there was intensive public education, vigilant surveillance in abattoirs and notification of all new human cases (Beard et al ., 2001). Incorporated into the control campaign, domestic dogs were denied access to offal and were dosed six weekly with arecoline and later six weekly with praziquantel (Beard et al ., 2001). Several key factors contributed to the eradication of E. granulosus from Tasmania: there are no dingoes in Tasmania, hence wildlife never became involved in transmission and consequently no wildlife reservoir developed; given that Tasmania is a small island stock movements could be controlled; the campaign was supported by state legislation; there was centralised recording of infection data from abattoirs and hospitals; and importantly there was good community support for the campaign because the public health advantages were obvious (Jenkins, 2005).

Although there has been an overall decline in echinococcosis in rural dogs on mainland Australia (Jenkins and Power, 1996), infection in certain focal areas remains a problem (Jenkins, 2005). Coproantigens of E. granulosus were detected in 29% of dogs from 95 farms in south eastern New South Wales and 17.5% from 43 farms in

Victoria (Jenkins et al ., 2006). Many owners of these infected dogs occasionally fed offal from domestic livestock and wildlife to their dogs and few owners wormed their dogs frequently enough to inhibit patent infections (Jenkins et al ., 2006). Although all of the owners had heard of hydatid disease and knew it was dangerous to feed sheep offal to dogs, most were unaware that macropods and feral pigs could also be infected with hydatid cysts (Jenkins et al ., 2006). Importantly it was found that some farmers thought that the term ‘offal’ only applied to everything below the diaphragm (lungs are the organs commonly infected with hydatid cysts in macropod marsupials) 144

(Jenkins et al ., 2006). An emerging concern is the encroachment of infected wildlife into urban areas and the infection of domestic dogs as a consequence of human activity (Thompson, 2001, Jenkins, 2006).

Human hydatid disease is no longer notifiable in Australia and estimates on the number of annual new cases are somewhere between 80-100 cases (Jenkins et al . unpublished data, cited in Jenkins, 2005). The majority of these new cases occur in the eastern states usually in rural areas associated with the Great Dividing Range (Jenkins,

2005), highlighting that this area is in need of a control program.

Important factors attributed to the continued prevalence of E. granulosus on mainland

Australia include the large and widespread wildlife reservoir, the lack of a national control campaign, a lack of routine recording of livestock hydatidosis in abattoirs, no notification of human cases, almost no information regarding the prevalence of infection in domestic dogs, the public is largely unaware of the existence and public health importance of E. granulosus and there is no State or Territory interest in legislative support (Jenkins, 2005).

The extent of E. granulosus on mainland Australia is unknown and it is imperative that a national surveillance program is initiated, supported and funded by the federal government in order to rectify the situation. The success achieved in Tasmania has highlighted the need for human hydatid disease to be made notifiable and hydatidosis in abattoirs to be recorded on a centralised database. This surveillance will identify areas where surveillance and control is needed in domestic dogs or dingoes/wild dogs as well as areas where an education program should be instigated. Farmers need to be informed that not only are livestock a source of infection to dogs but so too are macropods and educational material will need to clearly define what constitutes offal. 145

Targeted education is also needed in urban areas where dingoes/wild dogs have been sighted or where pet dogs may have access to macropods such as in urban/rural fringe areas. Furthermore, Jenkins (2005) has highlighted the public health risk for the general public visiting national parks where dingoes occur and perhaps in such areas signage and leaflets could be used to create public awareness.

7.5 Drug resistance

The first report of anthelmintic resistance in companion animals was recently documented. Kopp et al . (2007) reported pyrantel resistance in dogs experimentally infected with A. caninum in Brisbane, Australia. The current study revealed that the majority of dog owners complied with the prophylactic administration of anthelmintics at either 3 monthly or monthly intervals. An intensive interval treatment regime is likely to have produced selection pressure for anthelmintic resistance in populations of A. caninum (Kopp et al ., 2007b). This resistance reported in dogs infected with A. caninum parallels that reported in humans infected with A. duodenale , where, following a period of heavy pyrantel use, therapeutic efficacy was observed to fall (Reynoldson et al ., 1997).

Parasite resistance depends on a number of factors including the population dynamics of the host and parasite species, parasite genetics, parasite biology as well as the effects of the treatment (Thompson and Roberts, 2001; Wolstenholme et al ., 2004).

The population dynamics of domestic pets are different to those of livestock and consequently the progression of drug resistance is likely to be slower (Kopp et al .,

2007a). However, the dynamics in refuges and breeding facilities, where animals are regularly treated for helminths, is similar to the situation in livestock. Hypothetically, if resistance developed in such animals and then they were re-homed or sold, these

146 dogs and cats could harbour their own subpopulation of resistant worms. If one of these animals was subsequently introduced to an environment that had previously been free of larval helminths, the dog/cat would soon establish its own isolated parasite community, all of which would be resistant to chemotherapy (Thompson and

Roberts, 2001).

Selection pressure for resistance is largely affected by the degree of refugia

(Wolstenholme et al ., 2004). Refugia are subpopulations of parasites that remain untreated and they are important because the higher the proportion of the population in refugia, the slower the selection for resistance (Wolstenholme et al ., 2004). It has been suggested for livestock, that treatment should be confined to animals suffering from parasitism, and animals that can tolerate existing infections should be left untreated leaving an unselected refugium (Wolstenholme et al ., 2004). Given the zoonotic risk to pet owners and the potential of infection to other pets such an approach is inappropriate for companion animals. Refugia in pets is provided by the free-living and somatic reservoir stages of certain parasites as well as by the parasites infecting free-ranging, stray or feral dogs/cats and untreated pets. These free-ranging dogs/cats and untreated pets may defaecate in public parks and are an untreated reservoir host that may transmit infections to pets (Kopp et al ., 2007a).

Administration of a combination (given together as mixtures or sequentially) of anthelmintics from different classes to infected animals, is one way of reducing the rate of development of drug resistance (Wolstenholme et al ., 2004). Another way would be to reduce the frequency of drug administration and rely on client education as a means of preventing infection. The current study revealed that the majority of prophylactic anthelmintics administered to pets were broad-spectrum drug combinations. A potential adverse effect of such an approach is that certain drug 147 preparations maybe of sub-optimal efficacy and consequently lead to the development of drug resistance (Thompson and Roberts, 2001). For example when five commercially available canine anthelmintic products were tested on dogs naturally infected with A. caninum , three of these compounds reduced mean worm numbers by

76% or less (Hopkins et al ., 1998).

The results of the current study revealed that many pets were parasite free and that most pet owners, unaware of this situation, prophylactically administer broad spectrum anthelmintics to their pets at regular intervals at the recommendation of their veterinarian (who almost never undertakes a parasitological examination of the pets’ faeces). For the most part the threat of resistance would seem low given that many pets were parasite free, yet in the infected animals, administration of anthelmintics at frequent intervals without monitoring the efficacy of treatment could result in the development of resistance.

7. 6 Recommended approaches to control dog and cat GI parasites

It is time that the veterinary profession in Australia reassesses its approach to GI parasite control in companion animals. Treatments should be tailored according to parasitic risk factors and life cycles. In adult dogs and cats faecal samples should be examined prior to the administration of any anthelmintic, and the frequency of these examinations should be at the discretion of the veterinarian depending on the individual’s situation and exposure to parasitic risk factors. Puppies, kittens, pregnant and lactating animals are known to have a higher risk of infection and because it is difficult to determine parasitism in these animals, anthelmintics should be administered strategically. The importance of veterinarians educating all clients on means of minimising the risk of parasites to their pets and themselves cannot be

148 overemphasised and in the future should constitute the main means of parasite control/prevention.

149

APPENDIX 1 Brochure for pet owners

150

151

152

153

154

155

APPENDIX 2 Questionnaire for dog owners

If you own more than one dog please complete a column for each dog.

Please tick the appropriate box, circle the answer or fill in the answer in the space provided.

Name of Veterinary Practice

1. Your name: Your postcode:

2. Your dogs name: Dog 1 Dog 2 Dog 3

3. How old is your dog?

Dog 1 Dog 2 Dog 3 Years Years Years

4. What is the sex of your dog?

Dog 1 Dog 2 Dog 3 Male castrated (neutered) Male entire Female spayed (neutered) Female entire - lactating (pups on her) Female entire - pregnant Female entire - not pregnant or lactating Female unknown

5. Is your dog a purebred or a crossbred?

Dog 1 Dog 2 Dog 3 Purebred Crossbred (Go to Q7) Unsure (Go to Q7)

6. If your dog is a purebred dog, what breed is it?

Dog 1 Dog 2 Dog 3

7. How many dogs are in your household?

8. How many cats are in your household?

156

9. Approximately how often does your dog have contact (sniff, play, socialise) with dogs belonging to other households?

Dog 1 Dog 2 Dog 3 Daily 4-5 times a week 2-3 times a week Once a week Once a fortnight Never (Go to question 11) Other, please specify Unsure

10. If your dog has had contact with dogs from other households, approximately how many different dogs has it had contact with in the past week?

Dog 1 Dog 2 Dog 3

11. Approximately how many hours has your dog spent in an area used by other dogs in the past week (eg park)?

Dog 1 Dog 2 Dog 3

hours hours hours

12. Has your dog been treated for gastrointestinal (gut) worms, such as roundworms, in the last 12 months?

Dog 1 Dog 2 Dog 3 Yes No (Go to Q 17) Unsure (Go to Q 17)

13. Approximately how many times has your dog been wormed against gut worms in the past 12 months?

Dog 1 Dog 2 Dog 3

157

14. If you can remember the name(s) of the worming treatments used please write their names and the number of times these treatments were given in the last 12 months?

Number of times Number of times Number of times Name of worming used in the last 12 used in the last 12 used in the last 12 preparation months (dog 1) months (dog 2) months (dog 3)

15. Did you receive additional information about worms or worming with the treatment?

Yes (go to Question 16) No (go to Question 17) Unsure/can't remember (go to Question 17)

16. If you received additional information in what form was this?

Leaflet or brochure Verbal advice Poster Other, Please specify Unsure/can't remember

17. Where does your dog spend the majority of its time?

Dog 1 Dog 2 Dog 3 All the time in the house Mostly in the house Half inside, half outside Mostly outside All the time outside Unsure

18. Where does your dog MOST COMMONLY defaecate (pass its stools)?

In my home/yard on: Dog 1 Dog 2 Dog 3 Soil/sand/dirt Grass/lawn Garden bed Kennel/ dog run Litter tray

Other please specify:

Outside my yard at:

158

Park Beach Bush/scrub Street/verge Neighbours yard Other please specify:

Don't know/not sure

19. If your dog defaecates in your home/yard, approximately how often do you pick- up the faeces/droppings or empty the litter tray?

Daily 4-5 times a week 2-3 times a week once a week once a fortnight Never Other please specify Unsure

20. If you pick-up the faeces/droppings or empty the litter tray, what do you do with them?

Placed in a rubbish bin Put on or buried in the garden Put in compost heap/ compost tumbler Other, please specify:

Not applicable

21. In the past 12 months have you fed your dog meat (OTHER than processed meat from a can or pet sausage)? Dog 1 Dog 2 Dog 3 Yes No (go to Q 24)

22. Where did you obtain this meat? (please tick all appropriate answers)

Licensed butcher Supermarket Pet-food dealer Farm or rural property Other please specify Unsure/can't remember

159

23. What type of meat did you feed? (please tick all appropriate answers)

Fed Raw Fed after cooking Sheep/mutton/lamb Cattle/beef/veal Chicken Kangaroo Fish Pig/pork Rabbit Goat Horse Other please specify Unsure/can't remember

24. In the past 12 months have you fed your dog offal (liver, kidney, lungs)?

Dog 1 Dog 2 Dog 3 Yes No (go to Q 27)

25. What type of offal did you feed? (please tick all appropriate boxes)

Fed Raw Fed after cooking Sheep Cattle Chicken Kangaroo Pig Rabbit Goat Horse Other please specify Unsure/can't remember

26. Where did you obtain this offal? (please tick all appropriate boxes)

Licensed butcher Supermarket Pet-food dealer/supplier Farm or rural property Other please specify Unsure/can't remember

160

27. In the past 12 months, is it possible that your dog may have eaten from the carcass of a dead animal (eg sheep, pig, kangaroo, wild bird)?

Dog 1 Dog 2 Dog 3 Yes No/unsure (go to Q 29)

28. If yes, what type of animals do you think it might have eaten?

Dog 1 Dog 2 Dog 3 Sheep Cattle Chicken Kangaroo/wallaby Fish Pig Goat Horse Rabbit/hare Wild Bird Lizard Other please specify Unsure/can't remember

29. In the past 12 months have you taken your dog hunting?

Dog 1 Dog 2 Dog 3 Yes No (Go to Q 31)

30. If you have taken your dog hunting, what animals have you hunted? Please tick all appropriate boxes.

Dog 1 Dog 2 Dog 3 Birds Pigs Goats Kangaroos/Wallabies Rabbits/hares Other, please specify

31. In the past 12 months have you taken your dog bush walking, hiking or camping?

Dog 1 Dog 2 Dog 3 Yes No (Go to Q 33)

161

32. If yes, was the dog on a lead/leash all the time?

Dog 1 Dog 2 Dog 3 Yes No

33. In the past 12 months has your dog been to a farm, rural block or a place where livestock are kept?

Dog 1 Dog 2 Dog 3 Yes No

Parasite awareness

34. Are you aware of any internal parasites that dogs can transmit to humans?

Yes No (Go to Q 38)

35. If yes which of the following parasites do you think can be transmitted to humans from dogs?

Roundworms (Toxocara) Hookworms Flea tapeworm Hydatid tapeworm Heartworm Whipworms Giardia Cryptosporidium Other please specify

36. If you are aware that parasites can be caught from dogs, how can people become infected? (you may tick as many boxes as is necessary)

Patting/cuddling the dog Letting the dog lick you From dog saliva eg when throwing a ball for my dog Touching objects the dog has contacted (eg bedding) From picking up the dog faeces/stools Inhaling the dogs breath Contacting soil, lawn or plants with which the dog has also had contact Other please specify Unsure (Go to Q 38)

162

37. If you are aware that some parasites may be transmitted to people, where did you receive this information? (you may tick as many boxes as needed).

General practitioner, Medical doctor, Medical clinic Veterinarian, Veterinary clinic/hospital School, TAFE or University Breed society, Kennel club Pet shop Friends/family Health Department Television Radio Magazines Newspapers Produce store/Stock feed supplier With worming treatments Posters If so where? Other please specify Unsure/can't remember

163

Demographics

38. Please record the number of people in each age category living in your household?

Male Female Less than 5 years 5 to 10 years 11 to 15 years 16 to 25 years 26 to 35 years 36 to 45 years 46 to 55 years 56 to 65 years Older than 65 years

39. Please record the number of people who help look after the dog ie feeds the dog, walks the dog or decides on worming regime?

Male Female Less than 5 years 5 to 10 years 11 to 15 years 16 to 25 years 26 to 35 years 36 to 45 years 46 to 55 years 56 to 65 years Older than 65 years

This is the end of the questionnaire. Thank-you for taking the time to fill it out. Your veterinarian will contact you as soon as the results from your dog's sample are available.

164

APPENDIX 3 Questionnaire for cat owners

If you own more than one cat please complete a column for each cat.

Please tick the appropriate box, circle the answer or fill in the answer in the space provided.

Name of Veterinary Practice

1. Your name: Your postcode:

2. Your cats name: Cat 1 Cat 2 Cat 3

3. How old is your cat?

Cat 1 Cat 2 Cat 3 Years Years Years

4. What is the sex of your cat?

Cat 1 Cat 2 Cat 3 Male castrated (neutered) Male entire Female spayed (neutered) Female entire - lactating (kittens on her) Female entire - pregnant Female entire - not pregnant or lactating Female unknown

5. Is your cat purebred or crossbred?

Cat 1 Cat 2 Cat 3 Purebred Crossbred (Go to Q 7) Unsure (Go to Q 7)

6. If your cat is a purebred cat, what breed is it?

Cat 1 Cat 2 Cat 3

7. How many cats are in your household?

8. How many dogs are in your household?

165

9. Approximately how often does your cat have contact (sniff, play, socialise) with cats belonging to other households?

Cat 1 Cat 2 Cat 3 Daily 4-5 times a week 2-3 times a week once a week once a fortnight Never Other, please specify Unsure

10. If your cat has had contact with cats from other households, approximately how many different cats has it had contact with in the past week?

Cat 1 Cat 2 Cat 3

11. Approximately how many hours has your cat spent in an area used by other cats in the past week?

Cat 1 Cat 2 Cat 3

Hours hours hours

12. Has your cat been treated for gastrointestinal (gut) worms, such as roundworms, in the last 12 months?

Cat 1 Cat 2 Cat 3 Yes No Unsure

If NO/UNSURE please go to Question 17

13. Approximately how many times has your cat been wormed in the past 12 months?

Cat 1 Cat 2 Cat 3

166

14. If you can remember the name(s) of the worming treatments used, please write their names and the number of times you used these treatments in the last 12 months?

Number of times Number of times Number of times Name of worming used in the last 12 used in the last 12 used in the last 12 preparation months (cat 1) months (cat 2) months (cat 3)

15. Did you receive additional information about worms or worming with the treatment?

Yes No (go to Question 17) Unsure/can't remember (go to Question 17)

16. If you received additional information in what form was this?

Leaflet or brochure Verbal advice Poster Other, Please specify Unsure/can't remember

17. Where does your cat spend the majority of its time?

Cat 1 Cat 2 Cat 3 All the time in the house Mostly in the house Half inside, half outside Mostly outside All the time outside Unsure

18. Where does your cat MOST COMMONLY defaecate (pass its stools)?

In my home/yard on: Cat 1 Cat 2 Cat 3 Soil/sand/dirt Grass/lawn Garden bed Litter tray

Other please specify:

Don't know/not sure

167

19. Approximately how often do you pick-up the faeces/droppings or empty the litter?

Daily 4-5 times a week 2-3 times a week once a week once a fortnight Never Other please specify Unsure

20. If you pick-up the faeces/droppings or empty the litter tray, what do you do with them?

Placed in a rubbish bin Put on or buried in the garden Put in compost heap/ compost tumbler Other, please specify: Not applicable

21. In the past 12 months have you fed your cat meat (other than processed meat from a can)? Cat 1 Cat 2 Cat 3 Yes No (go to Q 24)

22. Where did you obtain this meat? (please tick all appropriate answers)

Licensed butcher Supermarket Pet-food dealer/supplier Farm or rural property Other please specify Unsure/can't remember

23. What type of meat did you feed? (please tick all appropriate answers)

Fed Raw Fed after cooking Sheep/mutton/lamb Cattle/beef/veal Chicken Kangaroo Fish Pig/pork Rabbit Goat Horse Other please specify Unsure/can't remember

168

24. In the past 12 months have you fed your cat offal (liver, kidney, lungs)?

Cat 1 Cat 2 Cat 3 Yes No (go to Q 27)

25. What type of offal did you feed? (please tick all appropriate boxes)

Fed Raw Fed after cooking Sheep Cattle Chicken Kangaroo Pig Rabbit Goat Horse Other please specify Unsure/can't remember

26. Where did you obtain this offal? (please tick all appropriate boxes)

Licensed butcher Supermarket Pet-food dealer/supplier Farm or rural property Other please specify Unsure/can't remember

27. In the past 12 months, is it possible that your cat may have eaten from the carcass of a dead animal (including a wild bird, lizard, mouse)?

Cat 1 Cat 2 Cat 3 Yes No/unsure (go to Q 29)

28. If yes, what type of animals do you think it might have eaten?

Cat 1 Cat 2 Cat 3 Wild Bird Lizard Mouse/Rat Frog Other please specify Unsure/can't remember

169

29. In the past 12 months has your cat wandered in the bush?

Cat 1 Cat 2 Cat 3 Yes No

30. In the past 12 months has your cat been to a farm, rural block or a place where livestock are kept?

Cat 1 Cat 2 Cat 3 Yes No

Parasite awareness

31. Are you aware of any internal parasites that cats can transmit to humans?

Yes No (Go to Q 35)

32. If yes which of the following parasites do you think can be transmitted to humans from cats?

Roundworms (Toxocara) Hookworms Flea tapeworm Heartworm Toxoplasma Giardia Cryptosporidium Other please specify

33. If you are aware that parasites can be caught from cats, how can people become infected? (you may tick as many boxes as is necessary)

Patting/cuddling the cat Letting the cat lick you Touching objects the cat has contacted (eg bedding) From picking up the cat faeces/stools Inhaling the cats breath Contacting soil, lawn or plants with which the cat has also had contact Other please specify Unsure (Go to Q 35)

170

34. If you are aware that some parasites may be transmitted to people, where did you receive this information? (You may tick as many boxes as needed).

General practitioner, Medical doctor, Medical clinic Veterinarian, Veterinary clinic/hospital Breed society, Kennel club Pet shop Friends/family Health Department Television Radio Magazines Newspapers Produce store/Stock feed supplier With worming treatments Posters If so where? Other please specify Unsure/can't remember

171

Demographics

35. Please record the number of people in each age category living in your household?

Male Female Less than 5 years 5 to 10 years 11 to 15 years 16 to 25 years 26 to 35 years 36 to 45 years 46 to 55 years 56 to 65 years Older than 65 years

36. Please record the number of people who look after the cat ie feeds the cat, or decides on worming regime?

Male Female Less than 5 years 5 to 10 years 11 to 15 years 16 to 25 years 26 to 35 years 36 to 45 years 46 to 55 years 56 to 65 years Older than 65 years

This is the end of the questionnaire. Thank-you for taking the time to fill it out. Your veterinarian will contact you as soon as the results from your cat's sample are available.

172

APPENDIX 4 Questionnaire for veterinarians

Practice Name : (not for distribution) Address

Please tick the appropriate box, circle the answer or write in the space provided.

DOGS On a 1 to 5 scale with 1 being Not a Problem and 5 being a Significant Problem, how much of a problem do you think the following parasites are in dogs in your practice area ? Not a A Significant Problem Problem

1. Roundworm 1 2 3 4 5 Don't Know (Toxocara)

2. Hookworm 1 2 3 4 5 Don't Know

3. Hydatids 1 2 3 4 5 Don't Know (Echinococcus)

4. Flea tapeworm 1 2 3 4 5 Don't Know (Dipylidium)

5. Tapeworm 1 2 3 4 5 Don't Know (eg Taenia/Spirometra)

6. Whipworm 1 2 3 4 5 Don't Know

7. Giardia 1 2 3 4 5 Don't Know

8. Cryptosporidium 1 2 3 4 5 Don't Know

173

On a 1 to 5 scale with 1 being Not Concerned and 5 being Very Much Concerned , how would you rate the following potential zoonotic hazards from dogs in your practice area ? Not Very much concerned concerned

9. Roundworm 1 2 3 4 5 Don't Know (Toxocara)

10. Hookworm 1 2 3 4 5 Don't Know

11. Hydatids 1 2 3 4 5 Don't Know (Echinococcus)

12. Flea tapeworm 1 2 3 4 5 Don't Know (Dipylidium)

13. Tapeworm 1 2 3 4 5 Don't Know (eg Taenia/Spirometra)

14. Whipworm 1 2 3 4 5 Don't Know

15. Giardia 1 2 3 4 5 Don't Know

16. Cryptosporidium 1 2 3 4 5 Don't Know

17. At what age do you usually recommend that pups be first treated for intestinal parasites?

Weeks

18. At what age do you usually recommend that pups be first treated for heartworm ? 19.

Months

174

20. Do you usually collect a stool sample from pups and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

21. Do you recommend a specific prophylactic program for puppies against intestinal worms?

YES NO (go to Q 26)

22. If yes please briefly outline your recommended program including the frequency you advocate.

Anthelmintic Age of dog Treatment regime

23. Do you recommend routine testing of nursing or pregnant bitches for intestinal worms?

Never Rarely Some of the time Most of the time Always

24. Do you usually collect a stool sample from nursing or pregnant bitches and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

25. Do you recommend a specific prophylactic program for nursing or pregnant bitches against intestinal worms?

YES NO (go to Q 30)

26. If so please briefly outline your recommended program.

Anthelmintic Treatment regime

175

27. If so please briefly outline your recommended program.

Anthelmintic Treatment regime

28. Do you recommend routine testing of mature dogs (other than nursing or pregnant bitches) for intestinal worms?

Never Rarely Some of the time Most of the time Always

29. Do you usually collect a stool sample from mature-aged dogs and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

30. Do you recommend a specific prophylactic program for mature aged dogs against intestinal worms?

YES NO (go to Q 34)

31. If so please briefly outline your recommended program.

Anthelmintic Treatment regime

32. What drug do you recommend most often for treatment or protection of dogs from:

Roundworms

Hookworms

Tapeworms

Heartworm

176

Cats

On a 1 to 5 scale with 1 being Not a Problem and 5 being a Significant Problem, how much of a problem are the following parasites in cats in your practice area ?

Not a A Significant Problem Problem

33. Roundworm 1 2 3 4 5 Don't Know (Toxocara)

34. Hookworm 1 2 3 4 5 Don't Know

35. Flea tapeworm 1 2 3 4 5 Don't Know (Dipylidium)

36. Tapeworm 1 2 3 4 5 Don't Know (eg Taenia/Spirometra)

37. Giardia 1 2 3 4 5 Don't Know

38. Cryptosporidium 1 2 3 4 5 Don't Know

39. Toxoplasma 1 2 3 4 5 Don't Know

177

On a 1 to 5 scale with 1 being Not Concerned and 5 being Very Much Concerned , how would you rate the following potential zoonotic hazards from cats in your practice area ?

Not Very much concerned concerned

40. Roundworm 1 2 3 4 5 Don't Know (Toxocara)

41. Hookworm 1 2 3 4 5 Don't Know

42. Heartworm 1 2 3 4 5 Don't Know

43. Flea tapeworm 1 2 3 4 5 Don't Know (Dipylidium)

44. Tapeworm 1 2 3 4 5 Don't Know (eg Taenia/Spirometra)

45. Giardia 1 2 3 4 5 Don't Know

46. Cryptosporidium 1 2 3 4 5 Don't Know

47. Neospora 1 2 3 4 5 Don't Know

48. Toxoplasma 1 2 3 4 5 Don't Know

49. At what age do you usually recommend that kittens be first treated for intestinal parasites?

Weeks

50. Do you usually collect a stool sample from kittens and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

178

51. Do you recommend a specific prophylactic program for kittens against worms?

YES NO (go to Q 57)

52. If so please briefly outline your recommended program.

Anthelmintic Age of cat Treatment regime

53. Do you recommend routine testing of nursing or pregnant queens for worms?

Never Rarely Some of the time Most of the time Always

54. Do you usually collect a stool sample from nursing or pregnant queens and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

55. Do you recommend a specific prophylactic program for queens against worms?

YES NO (go to Q 61)

56. If so please briefly outline your recommended program.

Anthelmintic Treatment regime

179

57. Do you recommend routine testing of mature cats (other than nursing or pregnant queens) for intestinal worms?

Never Rarely Some of the time Most of the time Always

58. Do you usually collect a stool sample from mature-aged cats and have it tested for intestinal parasites before anthelmintic treatment?

YES NO

59. Do you recommend a specific prophylactic program for mature aged cats against intestinal worms?

YES NO (go to Q65)

60. If so please briefly outline your recommended program.

Anthelmintic Treatment regime

61. What drug do you recommend most often for treatment or protection of cats from:

Roundworms

Hookworms

Tapeworms

Heartworm

180

ANTHELMINTHICS and INFORMATION

62. Do you ever discuss the potential zoonotic hazards of any of the following parasites with owners of dogs or cats?

Roundworms Hookworms Tapeworms Protozoa Yes Zoonotic potential discussed

No Zoonotic potential not discussed (go to Q68)

63. If you answered yes to any part of the previous question, how often do you discuss the potential zoonotic hazards with your clients? (Tick all appropriate boxes for the parasite you answered yes to).

Roundworms Hookworms Tapeworms Protozoa

Only when asked

Whenever worms are diagnosed in their pets

Routinely with new clients

Routinely with clients with puppies or kittens

Routinely with clients you know who have children

Routinely with all clients

64. What if anything do you recommend for the treatment of protozoa?

No treatment recommended

65. Where do you obtain new information on anthelmintics and dosing regimes?

Visiting Wholesale Drug Company Reps (eg Lyppards) Visiting Pharmaceutical Drug Company Reps (eg Bayer) Advertising material from drug companies

181

Professional/scientific/trade journals Conferences eg AVA annual conference Internet Other, Please specify:

66. If you provide information on parasites to owners, in what format is it? Please tick all appropriate boxes.

Verbal Pamphlets written by practice Pamphlets/brochures from drug companies Pamphlets/brochures from other veterinarians, AVA, or Health Dept Other, Please specify No information provided

67. If you provide information, does this include information on: Please tick all appropriate boxes.

Frequency of treatment? The lifecycle of the parasites? How the parasite is transmitted? The clinical signs of infection in the pet? The zoonotic risk of parasites?

That concludes the questionnaire. Thank-you for taking the time to complete it. Your answers will be valuable in determining the information provided to pet-owners by veterinary surgeons. A summary of the findings will be forwarded to you on completion of the study.

182

APPENDIX 5 Refuge data sheet

Name/ Murdoch label Age Sex/ Purebred/ Entire/ Wormed/ Animal Approx number (specify) neutered not housed how long has (2 other labels to be placed a) less than 1 month gender used to crossbred wormed separately/ the animal b) 1 month – identify on the yellow and white (at the in a group been at the 4month animal refuge) refuge capped tubes) c) 4 month – 1 year If wormed d) 1 year-7 years how many e) 7 years + days ago f) unsure 1

2

3

183

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