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    Application of diagnostic tools for optimised treatment and management of coccidiosis in kiwi (Apteryx spp.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Sciences at Massey University, Manawatū, New Zealand. EMBARGOED to 24 January 2027.
    (Massey University, 2024) Scheltema, Emma Margaret
    Coccidiosis, a disease caused by infection with the protozoan parasite, Eimeria spp., is currently the main limiting disease in captive and creche-reared kiwi and can cause significant morbidity and mortality in young birds. There are at least five species of Eimeria that infect kiwi, and they are usually present as a mixed species infection. These parasites cause damage to tissues of the intestine and occasionally to other organs including the liver, kidneys, lungs, and spleen. Infection is managed in captivity through husbandry and medication, primarily via therapeutic treatment with the anticoccidial, toltrazuril (Baycox®, Bayer, Leverkusen, Germany). However, there is some evidence that this drug is not always effective, and there has been no research to date into safe and effective alternatives. Thus, effective management of this disease in infected birds is limited by a lack of evidence-based therapeutic options. The overall objective of this study was to improve the detection and diagnosis of kiwi Eimeria spp. through the development of a molecular tool, that could then be applied to assist in identifying safe and effective prophylactic and/or therapeutic treatments for the control of coccidiosis in kiwi chicks. To achieve this, we developed a molecular diagnostic tool (qPCR) to measure the diversity of Eimeria species present in mixed infections in kiwi. This tool was applied to preserved tissue samples to identify the tissue specificity (in intestine, liver, kidney, lungs, and spleen) of different kiwi Eimeria species, which may have consequences for the success of drug treatment. Following the development of normal blood biochemical and haematological reference intervals in healthy kiwi chicks as a baseline, a safety and pharmacokinetic study of five anticoccidial drugs (amprolium 15mg/kg, decoquinate 0.5mg/kg, diclazuril 5mg/kg, trimethoprim-sulphamethoxazole 20:100mg/kg and toltrazuril 25 mg/kg) given as single doses to healthy kiwi chicks was carried out. We then trialled one of these drugs, diclazuril, given both prophylactically in-feed (0.5mg/kg q24h) and as a therapeutic treatment (5mg/kg single dose) in naturally infected kiwi in a field study at a captive-rearing centre. In combination with standard faecal oocyst counts, the molecular tool was used to assist in Eimeria species identification from faecal samples from treated and untreated birds. We were able to establish the presence of a single Eimeria species infecting extra-intestinal tissues, described here as Eimeria koka, and isolated another species Eimeria kiwii from the intestine. Baseline biochemical and haematological parameters and the safety and pharmacokinetics of four anticoccidial drugs were established in healthy three-to-four-week-old kiwi chicks. We were unable to determine the pharmacokinetics of one of the drugs, decoquinate. The use of diclazuril in-feed was well-tolerated by kiwi chicks and while there was no observed difference in body weight or feed intake between treated and untreated control chicks, diclazuril-treated birds shed significantly fewer oocysts than control chicks. A small number of birds were dosed therapeutically with diclazuril, and we observed a decrease in oocyst shedding equivalent to the toltrazuril-treated control group (standard management), however, more data is required to confirm this pattern. Not all kiwi Eimeria species were found to be equally susceptible to treatment with diclazuril, with changes in species composition observed in treated kiwi. The molecular assay developed in this study has a range of applications to address questions about kiwi Eimeria biology and control. We hypothesise that the newly described Eimeria koka is likely the only Eimeria species infecting kiwi that migrates extra-intestinally, to infect the kidneys, and under severe infections, probably disseminates to other organs. Further testing of the response of this species to drug treatment is required; it is likely more challenging to treat due to its location in the body. All drugs trialled in kiwi appear to be relatively safe at the given doses. Diclazuril appears to be partially effective in the prevention and therapeutic treatment of coccidiosis in kiwi. Further investigation into dose rate and period, and the addition of other safe and effective drugs to build a sustainable, rotational drug dosing scheme is highly recommended before the integration of any new treatments into captive management protocol. This study is the first to establish the safety and pharmacokinetics of anticoccidial drugs in kiwi, and to demonstrate the efficacy of diclazuril to treat coccidia infection in kiwi. The methods developed herein lay the foundation for establishing the safety and efficacy of other drugs for treating this disease and will ideally contribute towards the development of more sustainable coccidiosis management plans for kiwi. The identification of optimum treatments for coccidiosis in kiwi is of high importance for the ongoing maintenance of captive-reared kiwi health and welfare, and the success of this form of ex-situ conservation intervention.
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    The pathogenesis of pneumonia in sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 1975) Alley, Maurice Rewi
    The pathology of pneumonia in sheep in New Zealand is described in a study of over 400 naturally-occurring cases obtained from field and abattoir sources. The common forms of enzootic pneumonia consist of two distinct pathological and epidemiological entities; an acute pneumonia affecting sheep of all ages and a subacute or chronic, non-progressive pneumonia affecting lambs from approximately 3 to 10 months of age. Acute pneumonia is characterised by intense congestion, alveolar haemorrhage, fibrinous exudation and ventral consolidation of both lungs. Ultrastructurally the cellular exudate consists of a mixture of neutrophils, macrophages and detached alveolar epithelial cells with which bacteria are closely associated. Subacute and chronic pneumonia is characterised by varying degrees of dull red to grey consolidation of the anterior lobes. Ultrastructural studies reveal a variety of degenerative changes in the alveolar epithelium including several subcellular changes not previously recorded. Repair is by type II cell hyperplasia and this has been studied ultrastructurally and histochemically. Undifferentiated type II cells resembling those found in the foetal lamb and cells transitional between type II and type I have been observed. The significance of these findings in relation to the origin and dynamics of alveolar epithelial repair is discussed. The major factor underlying the pathological differences between acute and chronic pneumonia is considered to be the degree of damage to the alveolar epithelium which is universal in the former disease and less severe and localised in the latter. Experimental injury to the ovine lung produced by the endobronchial instillation of dilute (1%) nitric acid with India ink as a marker was studied at periods from 2 hours to 10 days after administration. Alveolar collapse and neutrophil infiltration were the earliest changes seen but few neutrophils remained after 3 days. Large macrophages which were active from 3 hours were joined by smaller macrophages which migrated from interstitial tissues from 12 hours until 3 days after administration. The ultrastructural changes observed in the alveolar epithelium were similar to those encountered in naturally-occurring pneumonia. Proliferation of Clara cells and type II cells was detected one day after administration and partial "epithelialization" of some alveoli at 5 days. There was complete loss of pulmonary surfactant from affected areas by 12 hours and return to normal activity was irregular. Parentally administered Paraquat and oral dosing with busulphan were also tested for their value as agents for producing experimental pulmonary injury in sheep. Maximum pulmonary involvement occurred at between 6 to 10 mg/Kg of Paraquat but death appeared to result from liver and kidney toxicity. Paraquat pre-treatment did not affect pulmonary resitance to endobronchially inoculated bacteria in pure or mixed cutures, however lesions similar in nature to those of acute enzootic pneumonia were produced by Staphylococcus aureus. No significant pulmonary effects were produced with busulphan at high dose rates. To investigate the bacterial flora of the respiratory tract of normal and pneumonic sheep, 184 normal sheep and 246 sheep aged 6 to 9 months with chronic or subacute pneumonia were examined at slaughter over a 2 year period. Pasteurella haemolytica was present in the nasal cavities of 73% of normal sheep and 78% of sheep with pneumonia, while Neisseria catarrhalis was also commonly isolated from both classes. Pneumonic lungs characterised by alveolar collapse yielded few bacteria whereas those in which cellular exudate predominated contained P. haemolytica in 75% of cases. In lungs with severe proliferative changes P. haemolytica was recovered in over 60% of cases and N. catarrhalis in 25 to 33%. The prevalence of Mycoplasma ovipneumoniae and Mycoplasma arginini was also investigated in the respiratory tract of normal and pneumonic 6 to 9-month-old sheep. Both organisms were ubiquitous in the nasal cavity but M. ovipneumoniae was recovered more frequently than M. arginini. The recovery rate and titre of M. ovipneumoniae in pneumonic lungs were substantially higher than in normal lungs and several proliferative histological features were found to be associated with these titres. Cellular exudation and epithelial hyperplasia were associated with combined high titres of M. ovipneumoniae and bacteria. Lymphoid hyperplasia and mucus secretion were associated with low bacterial titres. Transmission experiments with lung homogenate derived from cases of acute pneumonia succeeded in producing lesions similar to the natural disease when inoculated endobronchially into worm-free, housed lambs whereas cultures of P. haemolytica, M. arginini or pneumonic lung homogenised in medium containing antibiotic produced minimal or no effect. However, the excessive amount of inoculum and unnatural means of inoculation required suggested that host and environmental factors have a major role in the pathogenesis of the acute form of the natural disease. Serial transmission of subacute and chronic pneumonia was achieved by intranasal aerosol inoculation of lung homogenate derived from abattoir cases. The clinical signs and pathological lesions were similar in most respects to the naturally-occurring disease. The pathological development of the lesions was studied in a further transmission experiment in which 12 lambs were slaughtered sequentially from 2 to 12 days after inoculation. In studying the effect of various chemotherapeutic agents on the development of chronic pneumonia it was found that both ronidazole at 100 mg/Kg and oxytetracycline suppressed the development of the disease while tylosin and penicillin suppressed the development of the lesions without completely inhibiting the growth of micro-organisms. A controlled experiment to assess the effect of pneumonia transmission on weight gain produced a significant reduction in the weight gain of treated animals but there was no correlation between the weight gain of individuals and pneumonic lesions. It was presumed that the result was due to a transitory systemic effect immediately following inoculation. Intranasal inoculation of M. ovipneumoniae cultures produced lesions in 2 caesarian-derived lambs but inoculation of 9 worm-free housed lambs was unsuccessful. The balance of evidence indicates that pneumonia in sheep, as it occurs in this country, results from the interaction of host and environmental factors with infectious agents. In acute pneumonia, bacterial multiplication in alveoli, presumably damaged by systemic agents, is responsible for the destructive changes which occur. In chronic pneumonia bacteria from the nasal cavity actively contribute to the severity of the lesions but it is unlikely that they initiate the disease process. M. ovipneumoniae is also closely associated with the lesions of chronic pneumonia but further inoculation experiments and epidemiological studies are needed to define this organism's role more closely.