Thesis (Ph.D.)--Murdoch Unuiversity, 2009. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 180-189)

The Psittacidae is one of the most endangered families of birds in the world. Knowledge of its nutrition is important for understanding their survival and productivity in the wild, as well as for their adequate husbandry in captivity. Hand-rearing is a common practice for this group. However, research on their requirements is limited. Analysis of the crop content of chicks can provide new insights into psittacine nutrition, but it is limited by the small sizes of samples which can be obtained. We sampled the crops from free-living chicks of scarlet macaws and red-and-green macaws from southeastern Peru, Cuban parrots from the Bahamas, lilac-crowned parrots from northwestern Mexico, and thick-billed parrots from northern Mexico. The predicted metabolizable energy, protein, fat, minerals, profile of essential amino acids and profile of fatty acids of the crop samples, as well as from 15 commercial hand-rearing formulas, were analyzed and contrasted. Near Infrared Spectroscopy was shown to be a valid technique for the nondestructive, low cost prediction of a variety of nutritional attributes of crop samples as small as 0.5 g dry weight, expanding the possibilities of wild animal nutrition research. The diets of the five studied species presented remarkable similarities and common patterns. The predicted dietary metabolizable energy and fat concentrations were particularly similar among species, the thick-billed parrot being the one with the most unique nutrient profile. The fatty acid profile of the crop contents differed markedly among genera, with the thick-billed parrot closer to the macaws than to the parrots. In comparison with the crop samples, the hand feeding formulas presented lower fat, Mg, arginine, and valine concentrations. The wide variation in nutrients suggests that there is not yet a consensus among manufacturers concerning the correct nutrition for growing psittacines. It is suggested that a single formulation could be used to hand-rear macaws and parrots from half its nesting time to fledging, and further research should focus on their nutrition during the first half. Our results suggest that manufacturers should evaluate if increasing the concentrations of crude fat, Mg, arginine, and valine in commercial formulas enhances psittacine chick growth and health.

To enable assessment of recombinant BFDV capsid protein (recBFDVcap) for vaccination to protect against PBFD, commercially available lovebirds (Agapornis sp.) were tested for evidence of past and current BFDV infection using PCR, HI and HA to identify suitable BFDV-free birds in which to test the vaccine. During this attempt, it was found that lovebirds from commercial aviaries were endemically infected with BFDV with evidence of up to 100% prevalence of BFDV DNA in blood samples from individual birds over time. Such an approach was abandoned as unlikely to yield suitable numbers of naïve birds to conduct a BFDV vaccination trial. As commercially available lovebirds were considered to be a poor source of BFDV-free birds, wild caught cockatoo nestlings and eggs (long-billed corella; Cacatua tenuirostris and galah; Eolophus roseicapillus) were used to assess the efficacy of BFDV vaccination using baculovirus recombinant BFDV capsid. Eggs were artificially incubated and 3 eggs successfully hatched and 1 was successfully hand-reared. All nestlings were screened for BFDV DNA in blood using PCR upon arrival then on days 11, 18 and 25 and tested for anti-BFDV antibody on the day of arrival. All hatched birds were determined to be free of BFDV DNA and BFDV HI antibody in the peripheral blood throughout the hand rearing period and the flock was considered to be suitable for a BFDV vaccination trial. Corellas (n=13) were injected with 1 mL of vaccine containing 10 μg recBFDVcap on day 0 and 0.4 mL vaccine containing 66.8 μg recBFDVcap on day 11. All vaccinated corellas and 5 non-vaccinated control corellas were given 0.4 mL BFDV suspension (titre = log2 12 HAU/50 μL) intramuscularly and 0.1 mL orally 16 days after booster vaccination. Blood was collected periodically during the vaccination period and blood and feathers collected before and after BFDV administration. Testing included BFDV DNA detection by PCR and qRT PCR (on blood) as well as serum antibody detection by haemagglutination inhibition (HI) and BFDV DNA and antigen was detected by qRT PCR and haemagglutination (HA) (on feathers), respectively. Four of 97 blood samples collected from vaccinated birds post BFDV challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry (IHC). Non-vaccinated control corellas developed transient feather lesions and PCR, HI and HA test results consistent with PBFD. They were BFDV PCR positive for up to 41 days post-challenge and qRT PCR demonstrated reduced virus replication in vaccinated birds compared to non-vaccinated control birds. Thus, administration of recBFDVcap vaccine alone was found to incite an adaptive immune response in BFDV-free corellas that subsequently conferred protection against inoculation with BFDV. A commonly utilized method for excising blood dried on filter paper was proven to be of high risk of carryover contamination facilitated by a hole punch used for processing several samples. Therefore a practical method of avoiding carryover contamination was developed and used in the DNA testing procedures of the vaccination trial. Finally, the haematological characteristics of the above mentioned cockatoos were studied before and for 97 days after experimental infection with BFDV. It was found that the pre-challenge haematological values were similar between the vaccinated and non-vaccinated corellas. Most pre-challenge parameters were comparable to previously reported values of other cockatoos and psittacine birds. Significant differences were seen in both groups when comparing pre-challenge values with post challenge values for total and differential leukocyte concentrations, but PCV and TSP were not significantly affected by BFDV challenge.

Thesis (Ph.D.)--Murdoch University, 2007. / Thesis submitted to the Division of Health Sciences. Includes bibliographical references (p. 124-143).

Murdoch University (Ph.D.)--Murdoch University, 2008. / Contains three published journal articles at back of thesis. Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 196-231)

Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

Um componente da conservação de animais selvagens é a relocação de espécies comumente referida como projetos de soltura. Para que esta seja bem sucedida os candidatos do projeto devem estar livres de patógenos considerados de importância para a espécie. Segundo a Instrução Normativa 179 oficializada pelo IBAMA em 25/06/2008, determinou-se a realização de exames laboratoriais como medidas a se prevenir a introdução de agentes infecciosos no ambiente, e garantir a sobrevivência a longo prazo dos animais em questão. No Brasil encontra-se a maior quantidade em espécies de psitacídeos, e das 84 espécies, 13 são vulneráveis a criticamente ameaçadas de extinção. Diversos projetos de relocação de animais silvestres, incluindo vários já bem sucedidos com psitacídeos, vêm sido realizados em território nacional além dos existentes do exterior. O Papagaio-de-peito-roxo (Amazona vinacea) tem suas populações severamente afetadas, sendo classificada no estado de São Paulo como criticamente ameaçada e como ameaçada a nível mundial. Apesar das dificuldades para a conservação dos recursos naturais, existem remanescentes de habitat protegido e em regeneração que podem abrigar espécies que historicamente ocupavam estes locais, de maneira que projetos de reintrodução visando A.vinacea como espécie bandeira em São Paulo e em Santa Catarina, foram contatados para realizar a pesquisa sanitária prévia à soltura. Foram realizados suabes cloacais em amostragens seriadas de modo a identificar possíveis portadores para os agentes paramixovírus tipo 1, influenza tipo A, poxvírus aviário, coronavírus, Escherichia coli Enteropatogênica (EPEC), e Salmonella spp., em indivíduos da espécie confiscados do tráfico, através da reação em cadeia pela Polimerase (PCR), objetivando sequenciar os possíveis resultados positivos, discutindo a viabilidade e custos segundo as determinações da IN 179/2008. Amostras após a liberação também foram coletadas na forma de fezes obtidas no recinto de aclimatação e/ou ao redor dos comedouros de alimentação suplementar. Obtiveram-se um total de 151 amostras somadas em todas as coletas seriadas, sendo 103 amostras de suabes cloacais de aves ainda em cativeiro e 48 amostras de fezes não individualmente caracterizadas das aves soltas. Das amostras testadas para os agentes com potencial infeccioso obtiveram-se apenas resultados positivos para E.coli, totalizando 36 isolados, embora nenhum tenha sido caracterizado como EPEC. Observou-se uma tendência para maior detecção de E.coli nas amostragens iniciais em comparação com as finais, fato ligado principalmente às melhorias no manejo empregadas. A possibilidade de se comparar resultados em amostragens seriadas foi importante para uma avaliação mais segura quanto à sanidade dos animais envolvidos, auxiliando a determinar a seleção das aves, não havendo relatos de doenças imediatamente após a soltura ou no monitoramento a longo prazo. A baixa frequência de amostras positivas para os agentes que poderiam inviabilizar a liberação parece demonstrar que existe um exagero de que doenças representam um risco extremo impedindo projetos de relocação. Procedimentos de quarentena adequados e a realização de um mínimo de exames reduzem os riscos envolvidos, fato observado no presente estudo tanto em cativeiro como no processo de liberação e posteriormente. Para as instituições amostradas havia um limitado recurso anual que não seria capaz de pagar por exames individuais. Uma opção é a de testar amostras em pool para diminuir os custos, e caso haja positivos, procurar retestar para identificar os indivíduos. A baixa prevalência de positividade para os agentes com potencial infeccioso neste estudo, demonstra que amostras em pool podem ser uma alternativa econômica, caso o exame individual esteja fora de questão. Tendo em vista que para obter um mínimo de segurança no projeto de relocação no Brasil depende-se quase exclusivamente da iniciativa privada, convênios com universidades tornam-se não apenas uma necessidade, mas também uma oportunidade para troca em direção a um objetivo em comum e geração de conhecimento científico. / One component in the conservation of wild animals is the relocation of species, commonly referred as release projects. In order for this attempt to be successful the candidates must be clear of pathogens of significance for the species. According to the normative rule 179 established by the IBAMA in 25/06/2008, it was determined that a series of laboratorial exams should be performed in order to prevent the introduction of infectious agents in the environment and guarantee the long term survival of the animals. The largest number of psittacine species is found in Brazil accounting for 84 species, with 13 of these classified as vulnerable to critically endangered. Several relocation projects with wild animals, including several well succeeded with psittacines have been taking place on a national scale besides others being carried out around the world. The Vinaceous Amazon (Amazona vinacea) have had its populations severely affected being classified as critically endangered in the state of São Paulo and globally threatened. Although there are challenges to conserve natural resources, available remnants of protected and regenerating habitat can be found and could support species that historically inhabited these sites, hence reintroduction projects with A.vinacea as a flagship species in the state of São Paulo and Santa Catarina were contacted to perform a health survey previously to the releases. Cloacal swabs were taken in paired samplings in order to detect possible carriers for paramyxovirus typ1, influenza type A, avian poxvirus, coronavirus, Enteropathogenic Escherichia coli (EPEC), and Salmonella spp.; in individuals that were confiscated from the illegal trade employing the Polymerase Chain Reaction (PCR), aiming to sequence possible positive results and discussing the viability and costs according to the determination of the IN/179/2008. Fecal samples were also collected after the release in the acclimation flight and/or surrounding the supplemental feeders in the area while still adapting in the post-release. A total of 151 samples were obtained altogether with 103 of these being cloacal swabs of the birds still in captivity and 48 fecal samples non individually characterized of released birds. Out of the tested samples only E.coli yielded positive results with a total of 36 isolates, although none was characterized as EPEC. A tendency was observed for a higher detection of E.coli in the initial samplings compared to the final ones, a fact mainly connected husbandry improvements that were put in use. The possibility to compare results in paired samplings was important in order to obtain a safer evaluation concerning the health status of the animals, helping to determine the birds selection and no health problems reported neither immediately after the release nor on the long term monitoring. The low frequency of positive samples for the agents that could jeopardize a release seems to show that there is an exaggeration that diseases represent an extreme risk to the point of hampering relocation projects. Adequate quarantine procedures and performing minimum health examinations minimize the involved risks, a fact observed in this study both in captivity as well as in the release and post release process. For the studied institutions there was a limited annual budget that would not be capable to pay for individual exams. One option is to test pooled samples to lower associated costs, and if a positive is found, retest to identify the individuals. The low prevalence for the tested agents in this study show that pooled samples could be a viable alternative when individual exams are not feasible. Taking into account that to obtain a minimum in terms of safety for a relocation project in Brazil one is almost exclusively dependent on private parties, cooperation with universities are not only a necessity but also an opportunity for exchanges toward a common goal besides generating scientific knowledge.

Um componente da conservação de animais selvagens é a relocação de espécies comumente referida como projetos de soltura. Para que esta seja bem sucedida os candidatos do projeto devem estar livres de patógenos considerados de importância para a espécie. Segundo a Instrução Normativa 179 oficializada pelo IBAMA em 25/06/2008, determinou-se a realização de exames laboratoriais como medidas a se prevenir a introdução de agentes infecciosos no ambiente, e garantir a sobrevivência a longo prazo dos animais em questão. No Brasil encontra-se a maior quantidade em espécies de psitacídeos, e das 84 espécies, 13 são vulneráveis a criticamente ameaçadas de extinção. Diversos projetos de relocação de animais silvestres, incluindo vários já bem sucedidos com psitacídeos, vêm sido realizados em território nacional além dos existentes do exterior. O Papagaio-de-peito-roxo (Amazona vinacea) tem suas populações severamente afetadas, sendo classificada no estado de São Paulo como criticamente ameaçada e como ameaçada a nível mundial. Apesar das dificuldades para a conservação dos recursos naturais, existem remanescentes de habitat protegido e em regeneração que podem abrigar espécies que historicamente ocupavam estes locais, de maneira que projetos de reintrodução visando A.vinacea como espécie bandeira em São Paulo e em Santa Catarina, foram contatados para realizar a pesquisa sanitária prévia à soltura. Foram realizados suabes cloacais em amostragens seriadas de modo a identificar possíveis portadores para os agentes paramixovírus tipo 1, influenza tipo A, poxvírus aviário, coronavírus, Escherichia coli Enteropatogênica (EPEC), e Salmonella spp., em indivíduos da espécie confiscados do tráfico, através da reação em cadeia pela Polimerase (PCR), objetivando sequenciar os possíveis resultados positivos, discutindo a viabilidade e custos segundo as determinações da IN 179/2008. Amostras após a liberação também foram coletadas na forma de fezes obtidas no recinto de aclimatação e/ou ao redor dos comedouros de alimentação suplementar. Obtiveram-se um total de 151 amostras somadas em todas as coletas seriadas, sendo 103 amostras de suabes cloacais de aves ainda em cativeiro e 48 amostras de fezes não individualmente caracterizadas das aves soltas. Das amostras testadas para os agentes com potencial infeccioso obtiveram-se apenas resultados positivos para E.coli, totalizando 36 isolados, embora nenhum tenha sido caracterizado como EPEC. Observou-se uma tendência para maior detecção de E.coli nas amostragens iniciais em comparação com as finais, fato ligado principalmente às melhorias no manejo empregadas. A possibilidade de se comparar resultados em amostragens seriadas foi importante para uma avaliação mais segura quanto à sanidade dos animais envolvidos, auxiliando a determinar a seleção das aves, não havendo relatos de doenças imediatamente após a soltura ou no monitoramento a longo prazo. A baixa frequência de amostras positivas para os agentes que poderiam inviabilizar a liberação parece demonstrar que existe um exagero de que doenças representam um risco extremo impedindo projetos de relocação. Procedimentos de quarentena adequados e a realização de um mínimo de exames reduzem os riscos envolvidos, fato observado no presente estudo tanto em cativeiro como no processo de liberação e posteriormente. Para as instituições amostradas havia um limitado recurso anual que não seria capaz de pagar por exames individuais. Uma opção é a de testar amostras em pool para diminuir os custos, e caso haja positivos, procurar retestar para identificar os indivíduos. A baixa prevalência de positividade para os agentes com potencial infeccioso neste estudo, demonstra que amostras em pool podem ser uma alternativa econômica, caso o exame individual esteja fora de questão. Tendo em vista que para obter um mínimo de segurança no projeto de relocação no Brasil depende-se quase exclusivamente da iniciativa privada, convênios com universidades tornam-se não apenas uma necessidade, mas também uma oportunidade para troca em direção a um objetivo em comum e geração de conhecimento científico. / One component in the conservation of wild animals is the relocation of species, commonly referred as release projects. In order for this attempt to be successful the candidates must be clear of pathogens of significance for the species. According to the normative rule 179 established by the IBAMA in 25/06/2008, it was determined that a series of laboratorial exams should be performed in order to prevent the introduction of infectious agents in the environment and guarantee the long term survival of the animals. The largest number of psittacine species is found in Brazil accounting for 84 species, with 13 of these classified as vulnerable to critically endangered. Several relocation projects with wild animals, including several well succeeded with psittacines have been taking place on a national scale besides others being carried out around the world. The Vinaceous Amazon (Amazona vinacea) have had its populations severely affected being classified as critically endangered in the state of São Paulo and globally threatened. Although there are challenges to conserve natural resources, available remnants of protected and regenerating habitat can be found and could support species that historically inhabited these sites, hence reintroduction projects with A.vinacea as a flagship species in the state of São Paulo and Santa Catarina were contacted to perform a health survey previously to the releases. Cloacal swabs were taken in paired samplings in order to detect possible carriers for paramyxovirus typ1, influenza type A, avian poxvirus, coronavirus, Enteropathogenic Escherichia coli (EPEC), and Salmonella spp.; in individuals that were confiscated from the illegal trade employing the Polymerase Chain Reaction (PCR), aiming to sequence possible positive results and discussing the viability and costs according to the determination of the IN/179/2008. Fecal samples were also collected after the release in the acclimation flight and/or surrounding the supplemental feeders in the area while still adapting in the post-release. A total of 151 samples were obtained altogether with 103 of these being cloacal swabs of the birds still in captivity and 48 fecal samples non individually characterized of released birds. Out of the tested samples only E.coli yielded positive results with a total of 36 isolates, although none was characterized as EPEC. A tendency was observed for a higher detection of E.coli in the initial samplings compared to the final ones, a fact mainly connected husbandry improvements that were put in use. The possibility to compare results in paired samplings was important in order to obtain a safer evaluation concerning the health status of the animals, helping to determine the birds selection and no health problems reported neither immediately after the release nor on the long term monitoring. The low frequency of positive samples for the agents that could jeopardize a release seems to show that there is an exaggeration that diseases represent an extreme risk to the point of hampering relocation projects. Adequate quarantine procedures and performing minimum health examinations minimize the involved risks, a fact observed in this study both in captivity as well as in the release and post release process. For the studied institutions there was a limited annual budget that would not be capable to pay for individual exams. One option is to test pooled samples to lower associated costs, and if a positive is found, retest to identify the individuals. The low prevalence for the tested agents in this study show that pooled samples could be a viable alternative when individual exams are not feasible. Taking into account that to obtain a minimum in terms of safety for a relocation project in Brazil one is almost exclusively dependent on private parties, cooperation with universities are not only a necessity but also an opportunity for exchanges toward a common goal besides generating scientific knowledge.