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    Transmission of Campylobacter jejuni in broiler chickens : a thesis presented in partial (50%) fulfilment of the requirements for the degree of Masters in Veterinary Science in Veterinary Pathology and Public Health at Massey University, Palmerston North, New Zealand
    (Massey University, 2002) Ramabu, Solomon Stephen
    Investigations were carried out aimed at identifying potential sources of transmission of Campylobacter jejuni to broiler chickens, determining whether boots can mechanically transmit C. jejuni to susceptible chickens, determining the transmission rate of C. jejuni in broiler chickens, and assessing the effect of washing slaughterhouse fomites with chlorinated water on the level of C. jejuni contamination during the day. Bacteriological culture of samples taken in the premises of a broiler slaughterhouse showed that after washing and just before departure for depopulation of broiler farms, 75%, 58.33%, 47.22%, 50%, 54.29%, 66.67%, 31.25%, and 0% of pallets, crates, truck beds, truck wheels, drivers' boots, catchers' boots, forklift wheels, and tractor wheels respectively, were contaminated with C. jejuni. Therefore it was concluded that slaughterhouse personnel and fomites could potentially transmit C. jejuni during partial depopulation of broiler flocks. The level of C. jejuni in consecutive sample batches of fomites (n = 30) that were collected in six periods of one hour each day (hence six batches), between 0530 and 1230 hrs, over six days, was found to range from 44.83% in the lst batch and 66.67% in the 5th batch, indicating that washing fomites with pressurized chlorinated water makes no statistically significant difference (p>0.05) in the contamination level. The fomites sampled were pallets, crates, truck bed, truck wheels, and drivers' boots. Susceptible broiler chickens became colonized with C. jejuni after exposure to boots that had been worn in a pen with infected chickens demonstrating that boots can mechanically cause horizontal transmission of the bacterium. A transmission study conducted by introducing a bird infected with C. jejuni to 99 susceptible chickens demonstrated a rapid spread of the infection to all the birds within 48 hours and a transmission rate best represented by a simple epidemic curve. A transmission parameter beta (β), with a value of 2.1 x 10-3 ± 0.013 x 10-3 (mean ± standard error), was calculated by fitting the data into a simple deterministic epidemic model. The transmission rate in a population of 100 birds, predicted by this model, was very similar to that obtained in the experiment. Therefore, it was concluded that the transmission rate of C. jejuni in broilers resembles a simple epidemic.
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    Isolation and charaterisation of adenoviruses and reoviruses of domestic hens in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, New Zealand
    (Massey University, 1974) Green, Allan Fraser
    This investigation was undertaken to determine whether or not adenoviruses or reoviruses could be recovered from domestic hens in New Zealand. Using cultures derived from neonatal chicken kidney tissue 30 cytopathic agents were recovered from domestic hens. Electron microscope examination following staining by sodium phosphotungstate showed that 25 of these agents had the morphology of adenoviruses and the remaining 5 agents were morphologically indistinguishable from reoviruses. Some selected strains of adenoviruses and reoviruses were subjected to physico-chemical tests and it was found that both groups of agents were unaffected by chloroform and the replication of adenovirus strains, but not the reovirus, was inhibited by IDU. These results provide confirmatory evidence for the identification of these agents as avian adenoviruses and reoviruses. The avian adenoviruses like the analogous mammalian agents were found to have diminished thermostability in the presence of 1.OM Ca++. Cross-neutralisation tests showed that four serologically distinct adenovirus strains were recovered and 16 of the remaining adenovirus isolates were assigned to one or other of the four serotypes. All 5 reoviruses were assigned to one serological type on the basis of neutralisation tests. A preliminary serum survey for neutralising antibody to the 4 adenovirus serotypes showed that antibody to one or more of these viruses was present in a high proportion of flocks of domestic fowl in the Manawatu district of New Zealand and antibody to two of the serotypes was particularly common. This work represents the first report of the recovery of avian adenovirus and reovirus from domestic hens in New Zealand. However their clinical and pathological significance remain to bo investigated.
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    The aetiology and pathogenesis of avian inclusion body hepatitis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Virology, Massey University
    (Massey University, 1990) Saifuddin, Mohammed
    Naturally occurring inclusion body hepatitis (IBH) in broiler flocks in New Zealand, and the experimental disease were characterized by a sudden onset of illness resulting in up to 30% mortality and severe liver damage associated with the formation of intranuclear inclusion bodies in the hepatocytes. Other features were anaemia and atrophy of the bursa and thymus associated with lymphoid depletion. Serotype 8 avian adenoviruses (AAVs) were isolated from several affected broiler and one breeder flock. High titres of virus neutralizing (VN) antibodies were demonstrated in flocks which had recovered from the disease. By restriction endonuclease fingerprint analysis, two of the New Zealand isolates were found to be similar to each other and to the reference strain HVI, but markedly different from three Australian isolates of the same serotype. Fatal disease resembling IBH was reproduced in 30% of broiler chickens following oral administration of one of the local isolates. Immunosuppression was demonstrated in both natural and experimental infections. An enzyme-linked immunosorbent assay and an immunocytochemical technique were developed for the detection and quantification of adenoviral antigens in various chicken tissues. Both techniques detected less than 100 mean tissue culture infective doses per gram of infected tissue and a group-specific antigen common to the 12 serotypes of AAV. A study of the pathogenesis of IBH infection was conducted following oral administration of AAV. Virus first multiplied to a high titre in the intestinal organs and passed into the blood by way of the lymphatics. Viral antigens were subsequently detected in phagocytic cells in the liver and then in the hepatocytes. Extensive replication resulted in severe liver damage, with release of virus into the blood stream and spread to other organs. Recovery was associated with the appearance of VN antibody from 7 days post inoculation. Viral antigens were detected by ELISA directly in yolk and albumin of eggs derived from 50-60-week-old breeder flocks, although all birds had high titres of VN antibody in their blood. The inclusion bodies found in hepatocytes were characterized antigenically and ultrastructurally.
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    Epidemiological studies of highly pathogenic avian influenza in Vietnam : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University
    (Massey University, 2010) Minh, Phan Quang
    This thesis utilises data on highly pathogenic avian influenza (HPAI) subtype H5N1 from the Vietnamese national surveillance system and purpose-designed field studies to enhance the understanding of the epidemiological features of HPAI H5N1 in Vietnam. The findings obtained from the first study show that the presence of a HPAI H5N1 human case was associated with an increase in the likelihood of disease being detected in poultry one and four weeks later, indicating that the occurrence of clinical disease in poultry is not a useful predictor of subsequent human cases in the same locality. The analyses from the second study demonstrate that the epidemiology of HPAI H5N1 in poultry in Vietnam has changed over time and the infection transmission occurs by a combination of local and long-distance spread. The findings from a cross-sectional survey of management practices of itinerant grazing ducks suggest that surveillance strategies for this type of duck management should focus on both layer and larger flocks as they are more likely to be moved outside of their home district, facilitating long-distance disease spread. The results from a matched case-control study in poultry identify factors associated with the presence of HPAI H5N1 and provide evidence that disease control strategies should emphasise the reduction of household-level, rather than village-level, risks for disease. In the last study, spatio-temporal interaction of disease risk in poultry was observed within a distance of 10 kilometres and 12 days following the detected onset of clinical signs. Household-tohousehold infection rate within a commune was approximately 50 times greater than the household-to-household infection rate between communes. These findings show that the predominant mechanism of HPAI H5N1 infection transfer was local spread. The lessons learnt from the series of studies presented here should assist Vietnamese animal health authorities to implement the necessary systems and infrastructure that will allow novel and emerging disease syndromes to be investigated promptly and efficiently.
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    Biological and molecular characterisation and crystallisation of infectious bursal disease virus and its major capsid protein : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2001) Chai, Yew Fai
    Infectious bursal disease virus (IBDV) is prevalent in most of the poultry producing areas worldwide and causes severe economic losses resulting not only from clinical disease and mortality, but also from the immunosuppressive effect in subclinically infected flocks. The research on IBDV in this thesis is divided into two main parts. In the first part, studies were carried out on the IBDV inadvertently introduced into New Zealand in 1993. Prior to that date, the country had been free from the virus. IBDV was successfully isolated from seven flocks of subclinically infected and/or seronegative chickens in SPF embryonating eggs, adapted to cell culture and identified by EM, immunocytochemistry and RT-PCR test. To evaluate the efficacy of the serological method used in the screening programme of the IBDV eradication scheme, a study was undertaken to compare three diagnostic methods. The study demonstrated that serological testing is not a reliable method for the detection of IBDV infection in New Zealand broiler flocks because antibodies may not have developed to detectable levels by the time of slaughter. Histological examination of affected bursae allowed the demonstration of IBD-like lesions, but these needed to be differentiated from those caused by other agents. The immunocytochemistry test was able to detect early IBDV infection and provided a rapid, definitive diagnosis. Using the immunocytochemistry test to perform a longitudinal study of IBDV infection in a broiler and a layer farm, results showed the birds were infected as early as 6 to 7 days of age. The prevalence of IBDV infection was estimated to be 55% in the broiler flock. The results showed that the serological test had a sensitivity of 28.57% and a specificity of 73.68% for detecting the New Zealand IBDV strain infection. This indicated that there should be further evaluation of the use of serological testing as the sole method for the detection of IBDV infected farms in the current control scheme. An in-vivo pathogenicity study using IBDV derived from a bursal tissue homogenate and carried out in SPF chickens demonstrated the low virulence of the virus present in New Zealand. Molecular analysis of the hypervariable region of the VP2 gene of two 1BDV isolates obtained in 1997 and 1998 showed they are more closely related to attenuated strains than other strains. In all three phylogenetic analyses, using neighbour joining, parsimony and split decomposition, the NZ isolates are closely related to attenuated strain PBG98 and Cul but split away from Australia 002-73, variant E, classical and very virulent strains. Both results support the hypothesis that an attenuated strain of IBDV was inadvertently introduced into the New Zealand poultry population in 1993. In the second part of this thesis, studies on the structure of the IBDV virion and its major capsid protein were initiated by X-ray crystallography. The purification of IBDV for crystallisation and crystallisation trials are described. Several viral crystals were produced from the trials but only weak diffraction was obtained from these crystals. With the aim of studying the structure of the major capsid protein of IBDV (VP2) and investigating the major antigenic site on this capsid protein, the vp2 gene was cloned and expressed, the protein purified, and preliminary crystallisation trials performed. Recombinant VP2 was successfully expressed from a baculovirus expression system. The purification of the recombinant VP2 was also completed in the study and preliminary crystallisation screens determined several conditions favouring the production of crystals.
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    The epidemiology of Campylobacter jejuni in commercial broiler flocks in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Boxall, Naomi
    New Zealand maintains the highest incidence rate of human campylobacteriosis of the industrialized countries (334.2 cases per 100,000 in 2002), it accounts for more than 56% of all disease notifications in the country. New Zealand is unique globally, with a 'notification-based surveillance system for notifiable diseases that is complemented by laboratory reporting. In other countries (Australia, US, UK), the notification system is entirely laboratory based. Thus, the high incidence of Campylobacteriosis in humans may be related to the methods of reporting rather than the reality of the disease situation. However, the reason for such high incidence has not yet been fully elucidated, and several studies conducted in New Zealand and overseas have implicated the consumption of poultry meat as the main cause of human infections. The reduction or elimination of Campylobacter jejuni in the food chain, particularly from poultry meat products, is a major strategy in efforts to control campylobacteriosis. One approach to this is to prevent C. jejuni colonization of broiler chickens, This approach has been used to control Salmonella contamination of poultry, but the measures put in place for control of Salmonella have not controlled C jejuni. It is generally unknown how frequently C. jejuni colonizes commercial broiler chickens in New Zealand, or what could be done to prevent these infections from occurring. The present study was undertaken in order to describe some of the basic epidemiology of C. jejuni in commercial broiler flocks in New Zealand. The thesis is intended to further describe the epidemiology of colonisation of commercial broiler chickens by C. jejuni in NZ, and present possible risk factors that could be controlled in future to decrease the number of positive flocks of birds that are processed. The thesis set out to elucidate first the extent of C. jejuni colonisation of birds, flocks and farms while the birds were on the farm, having had minimal risk of exposure to Campylobacter spp., by sampling 15 birds in 80 flocks belonging to two companies prior to the first partial depopulation, an event during which the flock are exposed to potentially contaminated fomites and biosecurity levels are dropped, doors opened and personnel movements are extensive. The resulting prevalence estimates are 25.6% of farms, and 12.5% of sheds, are likely to be used to rear broiler chickens colonised with C. jejuni. When a positive flock is discovered, 76.9% of the birds are likely to be colonised with C. jejuni. These figures are results across the whole study population of farms and sheds, as there were no significant differences between prevalence estimates between companies. Following this prevalence estimation, a longitudinal study was conducted involving 12 sheds, to determine whether the environment or the birds were colonised with C. jejuni first. Although 12 sheds were observed every other day from day 14 to the end of the rearing period, it was determined that the birds were positive either first, or at the same time as the environment. Having said that, the sensitivity of the testing method for the environment was dubious, as there were instances where a shed that had positive samples collected on one occasion appeared negative the next, before returning a positive result on the third consecutive sampling occasion. A cross-sectional study of 810 flocks was undertaken to determine the most relevant risk factors for colonisation of the broiler chickens with C. jejuni. Because of the vertically integrated structure of the poultry industry, these 810 flocks corresponded to data collected from 77 farmers about their farms and the 219 sheds on those farms. The caeca from ten birds from each flock processed were pooled and examined for the presence of C. jejuni. These results were used to create a case definition, such that the flocks could be analysed with the questionnaire data, and different risk factors were seen in each season. More flocks reared for Company One were colonised by C. jejuni than for Company Two. Protective factors included having hard (i.e. gravel, asphalt or concrete) pathways to the growout houses, being near to another broiler farm, using the reticulated town water supply for the birds drinking water, using tunnel or crossflow shaped growout houses, using a Chore-TimeTM feed delivery system within the growout house and chlorinating the water supply to the birds (only in winter). The odds of raising flocks colonised with C jejuni increased if rodents were seen on the farm, if the growout houses were constructed with a concrete nib wall, if gas heaters were used during brooding, if cattle were farmed on the property, or if workers were employed on the farm. Sanitising the annex at least as frequently as once per run decreased the odds during summer, and tended to have a similar effect in other seasons. Chlorinating the water supply appeared to have a protective effect in only one season, though the trend appeared towards protection in the other seasons. The risk factor was validated by sampling the drinking water that broilers chickens had access to for the FAC to sec whether the levels that were present in the drinking water could have an effect on C. jejuni 11 sheds that were known to chlorinate the water were sampled to determine whether they met the drinking water standards for humans in NZ, or met the requirements presented by one of the companies involved. Only three sheds met the human drinking water standards for FAC, and two of these (one from each company) met Company Two's requirements. This thesis is for both regulatory and industry stakeholders to assist with developing risk management approaches to diminishing the number of C. jejuni positive flocks. Where management practices are altered, it is hoped that the efficacy of such practices be measured by examining the changes in the rates of C jejuni colonization within the industry
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    Epidemiological studies of Avian influenza viruses in Hong Kong : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Kung, Nina Yu-Hsin
    Eight studies of the epidemiological dynamics of avian influenza viruses were conducted on poultry in Hong Kong, with special focus on the movement of birds through the live poultry marketing system, and the implications for avian influenza transmission. The first involved analysis of virus isolation data from faecal samples obtained from cooperating stalls in live poultry markets in Hong Kong in a routine sampling program undertaken between 1999 and 2001. This showed that two subtypes of avian influenza viruses, H9N2 and H6N1, have become well established in the Hong Kong live poultry markets, especially in chickens (mainly H9N2) and quail (mainly H6N1). In addition, a wave of H5N1 virus infection occurred in 2001 after it had been absent through the earlier period of the study. The second study was an evaluation of long term changes in the marketing systems for live poultry in Hong Kong, their likely effects on avian influenza epidemiology, and documented evidence of the prevalence of various H subtypes of avian influenza in sampling undertaken during three periods from 1975 to 2001. The third project involved two cross-sectional studies and one longitudinal study on local quail farms in Hong Kong. Results showed that quail could act as a reservoir host for H9N2 and H6N1 subtype viruses, but each subtype differed in its epidemiology. H9 virus usually infected quail at around 10 days of age, whereas H6 infected quail at 15-20 days of age. At 35 days of age (usual market age in Hong Kong), a proportion of market quail were likely to be susceptible to H6 (50%) and H9 viruses (20%), and were able to transmit the virus in the markets. Due to these findings, the Hong Kong Government closed the quail farming operations and restricted the sale of quail in live poultry markets. A longitudinal study was conducted on three live poultry market stalls to assess the time from stall entry to sale, and evaluate the influence of this delay on avian influenza virus transmission to other poultry in the same stall. Participating stalls varied from high volume/rapid turnover to low volume/slow turnover. Turnover for tagged poultry was rapid, although some chickens became infected with H9 virus before sale. Birds which stayed longer, such as quail, became infected and maintained infection in the stall. As well as spread by direct contact and on typical fomites, keeping of pet roosters and re-use of washing water were identified as factors increasing the ability of avian influenza strains to persist in market stalls. An experimental replica of a market stall was created to study the transmission of low pathogenic avian influenza virus (H9N2) in a controlled environment, with different rates of population turnover and different immune status with respect to both H5 and H9 subtypes. Transmission was influenced by distance between birds, the proportion of birds carrying antibody to H9 and the rate of introduction of susceptible birds, but not by the use of H5N2 vaccine. In order to assess the effectiveness of temporary depopulation of stalls, a study was conducted to compare the prevalence of avian influenza virus and Newcastle disease virus before and shortly after the monthly rest day in live poultry markets in Hong Kong, by virus isolation. Prevalence of H9N2 avian influenza virus was reduced by the rest day, but Newcastle disease virus prevalence was unaffected. During the 2002 avian influenza H5N1 outbreak in local chicken farms, a case-control study was undertaken to identify risk factors that may have contributed to this outbreak. A questionnaire was administered by interview to collect the data for this study. Multivariate logistic regression models showed that movement of people and fomites from live poultry markets to farms were important influences on transmission, and that the live bird markets were the likely source of virus for farms. A spatial stochastic computer model was constructed to predict the spread of avian influenza virus in local chicken farms and live poultry markets in Hong Kong, and the effectiveness of control measures. The data used in model parameter setting was derived from the case-control study. The reference model produced an epidemic curve which was similar to the true epidemic curve in the 2002 outbreak. Control strategies such as rest day and vaccination were evaluated within the model, and found to produce results comparable with field experience. Results from these studies clarify various aspects of the epidemiological features and transmission dynamics of avian influenza viruses, and provide guidance on appropriate control and prevention strategies for highly pathogenic avian influenza viruses within poultry marketing systems in Asia.