Veterinary Science Collected Works - Theses

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    Studies on Intestinal Nematodes in Australian Thoroughbred Horses
    Abbas, Ghazanfar ( 2023-10)
    Gastrointestinal nematodes (GINs) are the most important parasites of equines as they pose a significant threat to equine health and wellbeing, particularly in younger and geriatric horses. Strongylids (cyathostomins and strongylins) and ascarids (Parascaris spp.) are the significant GINs of horses, with the former constituting more than 75% of the total parasite fauna. Heavy burdens of these parasites such as Parascaris spp. in young horses, can cause impaction and rupture of the small intestine while cyathostomins can infect all age groups of horses, with variable severity of cyathostominosis, particularly when encysted larvae emerge synchronously from the intestinal wall. The literature review (Chapter 1) identified various research gaps related to the epidemiology, diagnosis, efficacy of commonly used anthelmintics and control of GINs in Australian horses. Over the last 50 years, only a few studies have investigated the epidemiology of GINs of equines in different states of Australia which were either restricted to some regions or involved only a small number of horses. To address some knowledge gaps on the GINs in Australian horses, this thesis aimed to (i) establish baseline epidemiological data on GINs in Australian Thoroughbred horses, (ii) develop and/or employ more sensitive and advanced molecular tools for the detection of GINs in horses in epidemiological and drug efficacy studies, (iii) ascertain the efficacy of commonly used anthelmintics against significant intestinal nematodes of horses, and (iv) assess worm control practices used by Thoroughbred farm managers and equine veterinarians. The longitudinal (Chapter 2) and cross-sectional (Chapter 3) epidemiological studies conducted using coprological methods showed high prevalence and egg-shedding patterns of GINs in various age groups of horses. Climatic zone and age had the highest impact on faecal egg shedding, particularly in the Mediterranean climate, the autumn season, and young horses (i.e., yearlings). The polymerase chain reaction-directed next-generation sequencing (PCR-NGS) method uncovered the diversity of strongylid nematodes as 31 species were detected in both epidemiological surveys and their occurrence varied across various climatic zones, seasons and age groups of horses. Traditionally, the faecal floatation method has been used to diagnose eggs of Strongyloides westeri – a free-living parasitic nematode of newborn foals. In Chapter 4, following the detection of S. westeri eggs in the faeces of foals using microscopy, a PCR-based diagnostic method was established for the first-time using DNA extracted from the parasite eggs. This method will help conduct future molecular epidemiological studies on S. westeri and assess the efficacy of commonly used anthelmintics in foals. Chapter 5 showed the extent of anthelmintic resistance (AR) in cyathostomins and Parascaris spp. prevalent in Australian Thoroughbred horses. An apparent failure of the efficacy of a combination of anthelmintic drugs (i.e., oxfendazole (OFZ) and pyrantel (PYR)) was observed for the first time against Triodontophorus brevicauda – a species of large strongyles. Cyathostomins were resistant to multiple anthelmintics, including abamectin (ABM), ivermectin (IVM), moxidectin (MOX) and OFZ, whether used individually or in combination with other classes of anthelmintics, i.e., OFZ+PYR. Furthermore, where anthelmintics were effective 2 weeks post-treatment, egg reappearance periods (ERPs) were reduced to four and/or five weeks. The major cyathostomin species identified 2 weeks post-treatment were from the two genera, Cylicocyclus and Cylicostephanus while those 5 weeks post-treatment with MLs were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. Chapters 2 to 5 provided comprehensive information on the prevalence of GINs in Australian horses and resistance in ascarid and strongylid nematodes against commonly used anthelmintics. Subsequently, assessments of worm control practices surveys used by horse managers (Chapter 6) and veterinarians (Chapter 7) provided insights into their knowledge, aptitude and practices on GINs of horses, their diagnosis, treatment and control. Although both farm managers and veterinarians almost completely relied on anthelmintics to control GINs in horses, the latter seemed to use more targeted treatment strategies based on faecal egg count results. Multiple correspondence analyses used in Chapter 6 showed that the likelihood of suboptimal worm control practices on small farms (n = less than 50 horses) was greater than that of medium (n = 51-100) and large (n = above 100) farms. Furthermore, the findings highlighted a communication gap between veterinarians and horse managers. In conclusion, this thesis has contributed to addressing some key fundamental knowledge gaps on the GINs of Australian horses. Using conventional and advanced DNA-based diagnostic techniques, this thesis uncovered (i) the prevalence and egg-shedding patterns of GINs across various climatic zones during different seasons in various age groups of horses, (ii) the extent of AR against commonly used anthelmintics in ascarids and strongylid nematodes, and (iii) knowledge, aptitude and practices used by horse managers and veterinarians to control horse parasites. The novel findings of this thesis can pave the way for developing tailored guidelines for equine parasite control in Australia and globally.
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    The Likely Effect of Varroa destructor on Australian Honey Bee Populations
    Owen, Robert ( 2021)
    Varroa destructor, an invasive ectoparasitic mite of honey bees, is believed to be responsible for a large proportion of reported honey bee deaths globally. Australia remains the only Varroa-free country in the world with a beekeeping industry and horticulture sector reliant on Apis mellifera, the European honey bee. This thesis addresses issues essential to prevent the introduction of Varroa into Australia and better manage Varroa if an incursion (and then establishment) were to occur. The managed honey bee population in Australia is unusual, compared with other livestock industries, because a large sector of the industry (hobby beekeepers) are concentrated in locations where incursions of exotic diseases are likely to occur — urban areas adjacent to sea and international airports. For this reason, hobby beekeepers play an important role in early detection of honey bee diseases and limiting the spread of these diseases once an incursion has occurred. Hobby beekeeper networks need to be documented and appropriate communication and biosecurity resources developed for this sector of the industry to ensure roles and responsibilities are clearly defined (Chapter 3). A quantitative assessment of the Sugar Shake Team surveillance program for Varroa surveillance using scenario tree methods was made in Chapter 4 showing that if one of the 23,300 feral and managed apiariesin the Melbourne Metropolitan area were infested with Varroa there was only a 0.40% chance that it would be detected using the Sugar Shake Team surveillance program during one of the three or four surveillance events held each year. Reasons for low surveillance system sensitivity include poor sensitivity of the sugar shake method as a diagnostic test for the presence of Varroa in a colony and the relatively low frequency of testing of high-risk colonies in the greater Melbourne area. If the Sugar Shake Team program is to be used as a credible means for Varroa incursion detection the frequency of testing and the number of participants in the program needs to be increased. Research effort should be directed towards identifying more sensitive diagnostic tests to detect the presence of Varroa in honey bee colonies. If Varroa were to be introduced into Australia and there was a need to control the parasite using miticides, the cost per hive per year, if recommended controls were applied, would be in the order of AUD 51 (Q1 40; Q3 65) per hive per year (Chapter 5). In 2020 this represents a 17% reduction in net hive profitability. Assuming colony annual mortality rates attributable to Varroa ranging from 30% to 50% the percentage of apiary-year simulations where the cost of recommended Varroa controls was less than a suboptimal Varroa control strategy ranged from 32% to 43% (assuming a 30% Varroa attributable mortality rate) and 40% to 57% (assuming a 50% Varroa attributable mortality rate). While the use of suboptimal controls may be a pragmatic and rational economic choice for a beekeeper this represents a classic ‘tragedy of the commons’ situation whereby individual users, acting independently according to their own self interest, behave contrary to the common good of the industry. On the basis of these analyses, thought should be given to what changes can be made to the industry to make application of recommended Varroa controls cost effective for beekeepers. The first is to consider ways by which Varroa inspection and treatment times might be reduced. The second is to ensure that the cost of colony replacement is kept relatively high. A model was developed to simulate the introduction and spread of resistant Varroa Sensitive Hygienic (VSH) genetics into the managed honey bee population as a means for enhancing resistance of the feral population (Chapter 6). Introduction of VSH queens into the managed honeybee population had relatively little effect on the development of resistance in the feral population because the spread of Varroa resistance from the managed population to the feral population is slow compared with the rapid collapse of the feral population following Varroa the introduction of the mite. The only exception to this is that if the size of the resistant, managed population is large relative to the size of the feral population. This being the case there is scope for managed honey bee colonies in high risk areas to be seeded with Varroa-resistant queens to form a ‘barrier’ to limit the spread of the mite away from its point of entry into the country. If VSH genetics are to be used in this way as a means for either promoting Varroa resistance in the feral colony population or for iideveloping a Varroa resistance barrier around likely incursion sites (i.e., ports and international airports) it is essential that programs are established well in advance of a Varroa introduction.
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    Studies on gastrointestinal nematodes of alpacas in Australia
    Rashid, 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.
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    Molecular epidemiological investigations of Enterocytozoon bieneusi
    Zhang, 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.
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    Investigations into the critical aspects of the health and welfare of the bobby calves and dairy cows in Victorian dairy systems
    Pallab, 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.
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    The ecology and epidemiology of Rickettsia felis in Australia
    Teoh, 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.
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    Investigations of seed production potential of indigenous grassland forbs
    Delpratt, Cecil John ( 1999)
    This study investigated the potential for seed production, ex situ, from a range of forbs that were component species of grasslands and other grass-dominated plant communities in south-eastern Australia. Thirty-five species of native perennial forbs were collected from remnant grass-dominated plant communities in southern and western Victoria. All but one species (Tricoryne elatior, which did not germinate) were amenable to propagation and production in bark-based soil-less growing media in a variety of containers. Of 34 species, 29 (85%) flowered and produced seed within one year of propagation, and all species flowered and produced seed within 3 years. Of 20 species planted into field beds in autumn, 15 established successfully. The species generally were not amenable to spring planting in field beds. There was large variation between species in seed production potential due to differences in the number of flowers produced and the number of seeds produced per flower. Seasonal environmental effects on plant development and seed production were investigated in two studies. Firstly, Bulbine bulbosa and Craspedia variabilis were sown outdoors, in containers, at 12 sequential monthly intervals from 2/4/95 to 10/3/96. Both species established from all sowings but most flower and seed production occurred during spring and early summer, regardless of sowing date. For C. variabilis, sowings in January and February produced the most inflorescences per plant in the following spring. B. bulbosa plants produced visible buds approximately one month later than C. variabilis and were harvested 3 months later than equivalent sowings of C. variabilis. The second study investigated the potential for scheduling `out-of-season' production of seed. Seventeen species (that would normally be propagated in autumn) were sown in a greenhouse, and grown outdoors, on three occasions in late winter and spring (6/8/95, 3/9/95 and 1/10/95). Some plants of all but one species (Eryngium ovinum) flowered by April 1996, with 5 species exhibiting complete flowering in one or more sowings (Brachyscome dentata, Leptorhynchos tenuifolius, Velleia paradoxa, Wahlenbergia luteola, and W. stricto). Most species appeared to flower in response to interactions between cool (< 10 C) and warm (> 10 C) temperatures and to changes in photoperiod. It was concluded that to ensure synchronous flowering and, therefore, the potential for panmixis in out-breeding species, most species should be scheduled for flowering in their 'natural' flowering season. A method for improving the harvest efficiency of Bulbine bulbosa was investigated to replace the need for hand-harvesting of individual capsules. The yield, size and germination capacity of seed harvested from inflorescences that had been detached from the parent plant at a range of maturities, and dried at 20 degrees C, were compared to those of seed harvested from intact inflorescences. Seed yield was highest from intact inflorescences but daily harvests were required and harvests spanned a mean of 33 days per inflorescence, double the time needed for detached inflorescences to release all their seed. There was no significant difference between harvest methods in the number of seeds harvested per capsule, but there was a higher proportion of large seeds harvested from intact inflorescences. Germination was greater than 70% for all harvest treatments after 8 months of dry storage. Harvesting and drying inflorescences when one to three capsules had reached harvest maturity appeared to have the potential to increase harvest efficiency in B. bulbosa. The breeding system of Craspedia variabilis was examined in a greenhouse experiment that subjected inflorescences to one of three pollination treatments (none, hand self-pollination and hand cross-pollination). Cross-pollinated inflorescences produced an average of 301 achenes per capitulum, significantly more than either self-pollinated or non-pollinated inflorescences (19 and 15, respectively). It was concluded that C. variabilis is strongly out-breeding and largely self-incompatible. It was concluded that many perennial forb species were amenable to growth in containers but that seed production potential varied between species. For the genetic diversity sampled from remnant populations to be represented in seed produced in cultivated plants, the seed production system must take full account of the breeding system requirements and seasonal influences on flowering and seed production, for each species.
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