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ItemStudies on gastrointestinal nematodes of alpacas in AustraliaRashid, Mohammed Haronur ( 2019)The Australian alpaca industry has grown remarkably since the re-introduced of alpaca into the country in the 1980s. This relatively new and small industry faces various challenges that affect the overall production (fibre, meat etc.) of animals. For example, production losses and deaths associated with gastrointestinal nematodes (GINs) have become one of the major concerns for alpaca producers in Australia and elsewhere as there is a scarcity of reliable information about the epidemiology of GINs in alpacas, worm control practices used by alpaca farmers and the efficacy status of widely used unregistered anthelmintics. The present thesis aimed to (i) assess worm control practices used by Australian alpaca farmers, (ii) determine the epidemiology GINs and (iii) the efficacy of commonly used anthelmintics against GINs of alpacas in Australia. A comprehensive literature review along with seven results chapters and a general discussion chapter are included in this thesis. In Chapter 2, an online questionnaire survey was conducted to assess worm control practices used by Australian alpaca farmers. Results showed that more than half of the respondents perceived that GINs was an important health problem. Macrocyclic lactones (MLs) were the most commonly used anthelmintics in alpacas. Almost half of the respondents (47%) used anthelmintics at the dose rate recommended for sheep. The majority of alpaca farmers were unaware of pasture spelling and one-third of respondents had shared paddocks with ruminants. This study led to subsequent studies to understand the epidemiology of GINs of Australian alpacas using sensitive diagnostic methods and assess the efficacy of commonly used anthelmintics against GINs of Australian alpacas. In chapters 3 and 4, sensitive diagnostic methods were developed to determine the faecal eggs counts and identify nematode genus/species in the faeces of alpacas. In chapter 3, a newly developed diagnostic method, the FECPAKG2, was compared to a routinely used method, the McMaster technique, for the counting of GIN eggs in the faeces of alpacas. Data revealed moderate to good agreement between the two methods. This was the first study to assess the agreement of measurements between two methods for estimating nematode eggs in the faeces of alpacas. In Chapter 4, a molecular diagnostic tool based on multiplexed-tandem PCR (MT-PCR) was developed. This tool is faster and is capable of identifying common nematode genera/species of alpacas, including Camelostrongylus mentulatus which was not possible using traditional larval culture method. The epidemiology of GINs of alpacas in Australia was assessed using cross-sectional (Chapter 5) and longitudinal studies (Chapter 6). A range of GINs are prevalent in Australian alpacas with variable worm burdens in different climatic zones and seasons. The results of both studies were comparable. Both studies showed an overall prevalence of GINs in SACs from 61 – 66%. Weaners had the highest prevalence in both studies ranging from 73 - 80%. However, the pattern of prevalence was not same across the climatic zones. In cross-sectional study, the highest prevalence of GINs (77%) were observed in the summer rainfall zone, whereas in longitudinal study the winter rainfall zone had the highest prevalence (68%). In addition, a mixed-effects zero-inflated negative binomial (ZINB) regression model has been used to design parasite control interventions. To assess worm burden and the spectrum of GINs infecting Australian alpacas, 100 gastrointestinal tracts of alpacas were examined (Chapter 7). Results revealed a mean burden of 1,300 worms, with the highest burden of 29,000 worms. Nineteen different species of GINs were identified from Australian alpacas, including three camelid specific nematodes: Graphinema auchenia, Camelostrongylus mentulatus and Trichuris tenuis. Haemonchus contortus was the most prevalent nematode followed by C. mentulatus and Trichostrongylus spp. In Chapter 8, the efficacy of commonly used anthelmintics against GINs of alpacas was assessed. The faecal egg count reduction tests were conducted on 20 alpaca farms across the country. The results showed that a commercially available combination of levamisole, closantel, albendazole and abamectin was the most effective dewormer followed by single anthelmintic such as, monepantel, moxidectin, closantel, fenbendazole and ivermectin. Haemonchus spp. were the most commonly resistant nematodes. This was the first comprehensive study to investigate the efficacy of commonly used anthelmintics against GINs of alpacas. This study provides significant information on GINs of Australian alpacas. Results of this study advance our knowledge on the epidemiology and control of GINs and efficacy status of most commonly used anthelmintics which could be used to develop control strategies against GINs of alpacas in Australia.
ItemMolecular epidemiological investigations of Enterocytozoon bieneusiZhang, Yan ( 2019)Enterocytozoon bieneusi is a microsporidian microorganism that causes intestinal disease in animals including humans. Although E. bieneusi has been discovered and investigated in numerous countries around the world, at the commencement of this PhD project, there was no record of E. bieneusi in animals in Australia and no information on the prevalence and genetic make-up of E. bieneusi populations in this country. Thus, the present project explored E. bieneusi of a number of species of wild and domestic animals as well as humans in parts of Australia using a molecular approach, in order to improve our knowledge of the epidemiology of this microsporidian. Nested-PCR-based sequencing of the internal transcribed spacer (ITS) and/or small subunit (SSU) of nuclear ribosomal DNA was used for the detection of E. bieneusi DNA in faecal samples and the genotypic characterisation of this microbe. In the first instance, three species of wild deer (Cervus elaphus, Dama dama and Rusa unicolor; n = 610) and three species of wild marsupials (Macropus giganteus, Vombatus ursinus and Wallabia bicolor; n = 1365) in Melbourne's water catchment areas were investigated (Chapters 2 and 3). Here, E. bieneusi was detected in 4.1% of sambar deer and 1.4% of marsupials. Phylogenetic analyses of sequence data showed that genotypes D, J, MWC_d1, MWC_d2 and Type IV (in sambar deer) and MWC_m1 and NCF2 (in marsupials) clustered with E. bieneusi genotypes recorded previously in humans, indicating the zoonotic potential of these genotypes. Studies of cattle on farms located near Tarago reservoir (n = 471; Chapter 4) in Victoria, and alpacas in the states of New South Wales, Queensland, South Australia, Victoria and Western Australia (n = 81; Chapter 5) revealed relatively high prevalences of E. bieneusi in both cattle (10.4%) and alpacas (9.9%). The phylogenetic analysis of ITS sequence data revealed six genotypes (BEB4, I and J, TAR_fc1, TAR_fc2 and TAR_fc3) in cattle and three (ALP1, APL3 and P) in alpacas, all recognised to have zoonotic potential, indicating that these herbivores might act as reservoirs for human infection. Subsequently, a study of companion animals (cats and dogs; n = 514) identified genotype D, which is commonly detected in humans (Chapter 6). However, surprisingly, an investigation of humans (n = 605) in the states of Queensland and Western Australia (Chapter 7) did not identify the genotypes previously found in cats, dogs and wildlife in the state of Victoria, although, surprisingly, a known genotype (i.e., ALP1) recorded in farmed alpacas (Chapter 5) was identified in humans for the first time (Chapter 7). This latter finding raises questions about the transmissibility of this genotype from alpaca to humans. Overall, this thesis has recorded E. bieneusi, for the first time, in a variety of animals in Australia, and provides a first glimpse of the epidemiology of this microsporidian in this country (Chapter 8). The findings of this thesis indicate that the animals studied here, including eastern grey kangaroos, swamp wallabies and wombats; sambar deer, cattle and alpacas; and cats and dogs, can all carry E. bieneusi genotypes that might infect humans via water, food and/or the environment. To provide further evidence to support this proposal and much deeper insights, well-designed large-scale (temporal and spatial) epidemiological studies are required. Population genomics of E. bieneusi using advanced next-generation sequencing and informatics tools could significantly support this endeavour. Other future investigations might also, for example, attempt to establish in vitro culture and in vivo systems for E. bieneusi for controlled studies of the biology of distinct genotypes of this microbe as well as approaches for assessing the viability and infectivity of E. bieneusi and anti-infectives against this microbe. Clearly, we are only beginning to understand some aspects of this enigmatic pathogen - E. bieneusi.
ItemInvestigations into the critical aspects of the health and welfare of the bobby calves and dairy cows in Victorian dairy systemsPallab, Monoar Sayeed ( 2019)Animal welfare is becoming critical to the general public, farmers and dairy industry. In Australian dairy farming systems, maintenance of good welfare of large numbers of surplus non-replacement young male dairy calves (bobby calves) that go for slaughter at an early age of 5 to 10 days old is essential. Another major area of concern for the sustainable dairy industry in Australia is the active control measures against infectious pathogens that may cause severe diseases and production loss such as enteric pathogens in neonatal calves and Mycoplasma bovis (M. bovis) infection in cattle. This thesis examines the health and welfare conditions of bobby calves after transportation for slaughter and the potential of bobby calf blood samples to detect herd level M. bovis infection in dairy cattle in Victoria, Australia. Blood samples from bobby calves were collected at a commercial abattoir in Victoria after transportation and lairage to assess their welfare by examining plasma biochemical profile. A quarter of the calves had a failure of passive transfer with a variation of passive immune status depending on which region they came from suggesting colostrum management practices are not similar in all the farms. Very few calves experienced severe hypoglycaemia and dehydration before slaughter. However, most of the calves had higher plasma creatine kinase and lactate indicative of muscular fatigue. It is not clear from this study whether increased creatine kinase was also due to the muscular bruising in calves. Distance or duration of journey and lairage time had no significant effect on energy metabolites, hydration state or muscular fatigue in bobby calves before slaughter. Most of the calves at the time of slaughter showed no evidence of substantial physiological compromise, and there was no significant association either between the transport distance and plasma analytes nor between the total duration of transport and lairage in relation with plasma analytes. These results highlight that bobby calves can be well managed from the property of origin to abattoir under existing management system in Victoria without unduly compromising their welfare. The enteric pathogen E. coli K99 was the most common pathogen (37.4%) followed by bovine rotavirus (8.1%), Salmonella spp. (5.1%) and bovine coronavirus (2.6%) in the faeces of bobby calves after their transportation. Infected calves with the higher acquisition of passive immunity had lower amounts of bovine rotavirus (BRV), bovine coronavirus (BCV) and Salmonella spp. in faeces. Hypoglycaemia was associated with increased amounts of shedding of E. coli K99 and BRV in the faeces of infected calves. Increased distance of transportation was associated with a higher excretion of BRV only. Breed and sex had no influence on pathogen prevalence in the faeces. This study highlights that the prevalence of major enteric pathogens in bobby calves is minimal except E. coli K99 compared to previously reported prevalence of enteric pathogens in Australian dairy calves with diarrhoea, and higher acquisition of passive immunity may play an important role in lowering pathogen load in faeces of infected calves. The potential for bobby calf blood samples to be used to detect maternal antibody against M. bovis for the estimation of herd-level M. bovis prevalence in dairy cattle in Victoria was also assessed. Antibodies were detected using antibody capture ELISA. Sera were evaluated for adequate transfer of passive immunity before screening for M. bovis specific maternal antibody. All the M. bovis positive samples were detected from the sera with adequate passive immunity which was consistent with M. bovis specific antibody being transferred from cows to bobby calves. A proportion of 33.3% and 32.9% positive herds against M. bovis were detected in the northern and south-eastern dairy region respectively. These results indicate that M. bovis is a common pathogen in the major dairy regions in Victoria. This study also suggests that the collection of blood from bobby calves at the abattoir is convenient and could be used as a source of samples for M. bovis prevalence and surveillance study in Victoria, Australia.
ItemThe ecology and epidemiology of Rickettsia felis in AustraliaTeoh, Yen Thon ( 2018)Rickettsia felis is an emerging flea-borne zoonosis that is being increasingly recognised to contribute to unspecified malaise often referred to as fevers of unknown origins. The cat flea, Ctenocephalides felis, has been most widely accepted as the biological vector for R. felis. It is an adaptable ectoparasite that readily feeds on companion animals and opportunistically on other mammalian hosts. Its ability to persist in environments and on pets despite challenges from grooming, cleaning and prophylactic medications has led to the development of innumerable commercial preventatives designed to protect against infestation. The cat flea and its endosymbiont, R. felis, have managed to disseminate along with its canine and feline hosts across continents, presenting a potential risk to human health, Australia being of no exception. Despite the widespread presence of the cat flea and its promiscuous host-feeding behaviour, the contribution of R. felis to human morbidity, resulting loss of productivity and reduced quality of life in Australia remains largely unknown. Moreover, demographic, ecological and climatic risk factors for R. felis in Australia remain uninvestigated. This study sets out the foundation to address these knowledge gaps in Australia by i) retrospectively testing sera of patients previously testing positive for murine typhus for R. felis exposure; ii) determining the prevalence and associated demographic, occupational and geographical risk factors contributing to R. felis exposure in Australian veterinarians and; iii) determining the role of rodents as peri-domestic reservoirs for R. felis, and; iv) determining the geographical and climatic risks contributing to R. felis infestation rates in cat fleas in eastern coastal Australia. Historically, a specific diagnosis of flea-borne spotted fever (FBSF) was limited, owing to immunogenic cross-reactivity with Rickettsia typhi. Thus patients who had been exposed to R. felis were not able to be specifically tested. Of 49 patient sera previously testing antibody positive for murine typhus at the Australian Rickettsial Reference Laboratory, Geelong, 14 patients yielded specific antibodies to R. felis, seven yielded specific antibodies to R. typhi, while 28 patients were classified as indeterminate (with titres reacting between two-fold across the two organisms). Of R. felis positive patients, 5 demonstrated seroconversion, indicating a recent infection that was likely contributing to clinical signs observed by their referring doctor. Despite a cluster of five individual FBSF cases being reported for the first time in Australia in 2009, it is clear that other FBSF cases have remained undiagnosed or misdiagnosed, advocating the need for medical practitioners to obtain relevant history on patient’s exposure to domestic pets and their fleas.The second study sought to address the prevalence and associated risk factors for R. felis exposure in a high-occupational risk group, veterinarians. Of 131 veterinarians tested across Australia, 16.0% were found specifically seroreactive to R. felis. Older veterinarians were significantly protected from exposure (OR=0.752,p=0.04, 95% CI=0.579−0.975), as were those that recommended flea treatment to their clients (OR=0.611,p=0.044, 95% CI=0.38−0.982). Surprisingly, participants from the cooler south-eastern states of Victoria and Tasmania were trending toward a higher odds of exposure compared with other, warmer states (OR=1.381,p=0.075, 95% CI = 0.973−1.96). This was at odds with preconceptions on the ecology of ectoparasites; warmer subtropical and tropical regions were associated with higher environmental flea burdens. Moreover, this study demonstrated the high degree of exposure of R. felis in veterinarians, suggesting incidence of FBSF may be underrepresented in Australia. For the third study, fleas collected from client-owned dogs and cats in different climatic zones along the east coast of Australia were screened for R. felis, to determine infection rates of R.felis URRWXCal2 in C. felis felis. Infection rates of 6.7%, 13.2% and 15.5% were obtained by real-time PCR (qPCR) in tropical, subtropical and temperate regions, respectively. Univariate analysis indicated that fleas from toy/small breed dogs were less likely to be harbouring R. felis (p=0.033), as were Domestic Medium Hair (DMH) and pedigree-breed cats (p=0.0002 and p=0.043 respectively). Cooler minimum temperature ranges of between 15 to 20°C and between 8 to 15°C increased the odds of R. felis positivity in fleas, as did a constrained maximum temperature range of between 27 to 30°C on multivariable analysis. In conclusion this study demonstrated that environmental conditions such as temperature play a part in influencing R. felis survival and infectivity within its flea host, which suggests that public health risk mitigation measures need to consider regional differences to adequately comprehend transmission risk of flea-borne spotted fever. Finally, the involvement of peri-domestic animals in the life cycle of R. felis was investigated. Within urban environments, movement of companion animals is limited by legislation and animal control initiatives. Rodents are able host the cat flea vector, but their participation in perpetuating the life cycle of R. felis hasn’t been conclusively demonstrated. Brain and spleen tissue collected from 256 native and introduced rats (Rattus fuscipes, Rattus norvegicus and Rattus rattus) in south-east Queensland were tested for rickettsiae by qPCR, with none detectably positive, suggesting that they do not play a significant part in maintaining R. felis. This series of studies represents an in-depth look at the ecology of R. felis in Australia, and provides an insight into characteristics that makes it such a successful but often overlooked organism. Awareness amongst the public, medical practitioners and veterinarians is currently poorly developed, and diagnosis of the condition has suffered as a result.