Neste trabalho são apresentados resultados relacionados a estudos sobre o comportamento eletroquímico do lactato e do peróxido de hidrogênio em diversos eletrodos em meio de diferentes pHs utilizando voltametria cíclica. Também foi investigado o comportamento eletroquímico do peróxido de hidrogênio em eletrodos modificados com filmes de hexacianoferratos utilizando eletrodos de platina e carbono vítreo. A vantagem do uso do CTAB na modificação da superfície de carbono vítreo com Azul da Prússia foi confirmada no que se refere à melhora da sensibilidade e da estabilidade das medidas. Numa etapa posterior desenvolveu-se um biossensor para a determinação de lactato pelo monitoramento de peróxido de hidrogênio produzido na reação catalisada de lactato com oxigênio na presença da enzima lactato oxidase. Nessa etapa, o trabalho consistiu em imobilizar a enzima lactato oxidase na superfície do eletrodo, previamente, modificado com Azul da Prússia, com o auxílio de Nafion®. Após a construção do biossensor e a otimização das condições de análise (pH, quantidade de enzima, volume da amostra e vazão) para obtenção de maior sinal analítico no desenvolvimento do método para a determinação de lactato por análise em fluxo, averiguou-se a repetibilidade das medidas (Clactato = 0,28 mmol L-1) obtendo-se um desvio padrão de 2,2% para 18 repetições. A freqüência analítica foi estimada em 160 injeções por hora com limite de detecção de 0,84 µmol L-1 e linearidade até 0,28 mmol L-1 de lactato. O biossensor foi aplicado na quantificação de lactato em amostras alimentares (cervejas alcoólicas e não alcoólicas) e biológicas (sangue liofilizado e recém coletado). Por fim, realizaram-se estudos envolvendo a variação da concentração de lactato sanguíneo em função da intensidade de atividade esportiva. Os resultados obtidos pelo método proposto foram comparados com aqueles oriundos do uso de método de referência. / Results on the investigation of the electrochemical behavior of lactate and hydrogen peroxide at various electrodic surfaces at different pHs using cyclic voltammetry are presented. Experiments were also carried out with platinum and glassy carbon electrodes modified with hexacyanoferrate films. The advantage of using CTAB in the electrodeposition step of Prussian Blue films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of measurements. The immobilization of lactate oxidase onto glassy carbon electrodes modified with a Prussian Blue layer using Nafion® was performed to fabricate a biosensor for lactate. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 mmol L-1 lactate with a limit of detection of 0.84 µmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The analytical frequency was calculated as 160 injections h-1. The usefulness of the method was demonstrated by determining lactate in beer (alcoholic and nonalcoholic beers) and blood samples (lyophilized and freshly collected). Finally, the influence of physical exercise on the blood lactate level was studied. Results obtained by using the proposed amperometric detector compared well with the reference method.

Barramundi (Lates calcarifer) aquaculture is expanding throughout Australia and the Asia-Pacific region. The Department of Primary Industries, Queensland have estimated that the production from this industry could reach $30 million per annum in Australia by 2010. However, current production is severely impeded by outbreaks of Streptococcus iniae, which causes a fatal septicaemia in barramundi. S. iniae is a Gram positive bacterium which infects both humans and fish and was first reported in Australia in the 1980s in Queensland, but has rapidly disseminated to other states in Australia (Western Australia, Northern Territory, South Australia). Globally, there appears to be little geographical restriction to the distribution of S. iniae and infection occurs in temperate, sub-tropical and tropical, marine and fresh water fish with no evidence of species specificity. Outbreaks have been reported in North America, Middle East, Europe and Asia-Pacific, including Australia, Indonesia, Malaysia, Korea, China, Taiwan and Japan. Understanding distribution and spread of S. iniae is confounded by a number of factors. Firstly, identification of S. iniae is not straightforward, thus isolates often remain ‘unidentified’, as this bacterium is not included in commercial databases. In other cases it is misidentified as other bacteria such as S.uberis, S. dysgalactiae subsp. equisimilis and S. anginosus. Furthermore, variability in phenotypic traits has led to difficulty in identifying isolates using standard commercial diagnostic kits. Additionally, there is tangible evidence of geographic diversity and endemism with strain variability having been reported from fish isolates in Japan, USA and Israel, and in human isolates from Canada, USA and SE Asia. Understanding strain diversity amongst S. iniae is critically important in terms of managing the disease. Ability to track routes of distribution of the pathogen in imported fish, including ornamentals and food fish has implications for better biosecurity. Perhaps most importantly, strain diversity has been reported as a cause of vaccine failure in trout in Israel and in barramundi in Northern Territory, Australia. To date, very little information exists on strain diversity in S. iniae and no research has been conducted on the diversity amongst Australian isolates within the barramundi industry. The aim of this thesis is to develop reliable methods for identification of differing strains of S. iniae and to investigate antigenic diversity in order to better inform both vaccine design and biosecurity procedures with which to manage this important disease in Australia and globally. To achieve this, a collection of more than 100 isolates from Australia and throughout the world has been created and stored at the University of Queensland. In the first chapter of my thesis, routine confirmatory diagnosis using amplification of the lactate oxidase gene was performed to support biochemical and physiological identification provided by the supplying laboratories and veterinarians. During this initial screen, two important discoveries were made. Firstly, S. iniae isolates can be divided into two groups based on the different sizes of PCR product obtained, 869 bp (now named Type 1) and 921 bp (now named Type 2). This difference was only found in isolates from Northern Territory, Australia. In light of this, identity was further confirmed by the results of partial sequencing of the 16S rRNA gene with the 530F primer and submission to the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST), which returned identities of 100% to S. iniae ATCC 29178. Sequence analysis of the lctO gene in isolates representing both the normal (lctO type 1) and higher molecular weight (lctO type 2) revealed that there is an insertion of 51 bp of repeat sequence in lctO type 2. Apart from the insertion sequence found in the 3' end of the gene in some isolates, three nucleotides in positions 211-213, not previously detected when the gene was described previously, resulted in an inserted valine residue in the translated product from all isolates. I also note an apparent error in the primary sequence and translation of the GenBank sequence (Y07622). This is likely to be due to an inserted C nucleotide at position 1148 at the far 3’ end of the gene sequence (inside the LOX-1, LOX-2 priming region) that has altered the reading frame. This means that the expected PCR product size of 870 bp is incorrect and is actually 869 bp. To determine the phenotypic relevance of the variation in lox gene product size, the lactate oxidase enzyme was extracted from cell lysates and assayed for activity. The two variant genes were each cloned and expressed in E. coli. Lactate oxidase enzyme activity also showed that there were differences in enzyme activity between the two gene products with strains expressing the higher molecular weight enzyme variant exhibiting higher enzyme activity. This suggests that positive selection may apply in favour of the larger gene in situations where lactate is the most readily available carbon source. However, no variation was detected in the lactate permease gene lctP, for any of the strains analysed. Whilst there was no difference in the Minimum Inhibitory Concentration Test (MIC) using different concentration of lactate there were differences detected in the growth rate of QMA0165 and QMA0177. Significant inhibition on growth rate of QMA0165 was detected with a 0.3% and 0.5% of lactate while there was no significant inhibition in QMA0177 with the same concentration. Prediction on three dimensional protein structure using PyMol based on Aerococcus viridans 3-D protein structure showed that there was an additional loop in lctO type 2 which suggested that it might play a role in enhancing enzyme activity of the binding site. The environment in barramundi farms in Northern Territory where the lctO type 2 isolates were isolated has 9 metre tides resulting in water flows in excess of 3km/h. It is likely that the resulting enforced swimming of the fish host has led to selection and maintenance of a gene encoding the higher efficiency enzyme in S. iniae. As diversity has led to reported vaccine failure in Israel, and antigenic diversity has been recorded in Japan and in isolates from the USA, the second data chapter of this thesis explores surface antigenic diversity of Australian S. iniae isolates from barramundi using a whole cell ELISA using a suite of antibodies raised in barramundi against four S. inaie isolates from differing habitats (freshwater and marine) and states (Western Australia and Queensland) in Australia. Forty-one isolates predominantly from farmed fish throughout Australia between 1995 and 2006 were serotyped and compared with reference isolates from the USA and Canada. Multiple serotypes were identified using polyclonal sera raised in barramundi against four different Australian S. iniae isolates i.e. anti QMA0072 (Queensland), anti QMA0074 (Queensland), anti QMA0083 (Western Australia) and anti QMA0087 (Western Australia). Different serotypes were often isolated from the same sites either simultaneously or within short time periods, indicating potential coexistence of multiple isolates in a particular geographic location or habitat. Electrophoretic profiles of whole cell proteins and integral membrane proteins were similar amongst isolates when analysed by SDS- PAGE, regardless of serotype. The results presented here suggest that surface serotypic variability of S. iniae is complex and multifactorial involving capsular carbohydrate and some surface proteins. As raising consistent antiserum in barramundi is almost impossible, and rabbit antisera invariably recognise more epitopes than teleost fish, a more consistent molecular method of typing was investigated in the third data chapter. In this chapter, the whole genome of S. iniae was digested using SmaI and separated using Pulsed Field Gel Electrophoresis (PFGE). Twenty four isolates representing different geographical origin and host were analysed using this method. Reference isolates from dolphins, fish and humans were obtained from the Centers for Disease Control for comparison. PFGE profiles indicated at least 4 distinct groups amongst the Australian isolates, but these did not correlate with surface serotype. Interestingly, whilst there have been no reports of human cases of S. iniae infection in Australia, many of the isolates examined had closely related PFGE profile with reference human isolates from USA and Canada, but were markedly different from the type isolates isolated from dolphins. One of the major difficulties associated with PFGE is between lab variability, hence the requirement for inclusion of large numbers of reference isolates on each gel. Recently, multilocus sequence typing (MLST) has been developed for epidemiological studies in a variety of human pathogens. MLST is based on sequencing of 8 ubiquitous housekeeping genes, genes which evolve slowly, allowing clustering of isolates. As sequencing is consistent between laboratories, results can be posted on a website and compared internationally without requiring transfer of strains overseas. The fourth data chapter in my thesis develops for the first time an MLST scheme for S. iniae. Primers for eight housekeeping genes were designed and annealing temperature for amplification were optimized. The selected housekeeping genes were: adhP (Alcohol dehydrogenase), pheS (Phenylalanyl tRNA syhthetase), atr (Amino acid transporter), glnA (Glutamine synthetase), sdhA (Serine dehydrogenase), glcK (Glucose kinase) and tkt (Transketolase). However, glnA was dropped from the analysis because of the inconsistent PCR product. Thirty seven isolates were selected representing the Australian isolates and other international isolates from United States, Canada, Israel, Thailand, Reunion Island with different hosts i.e. Amazon freshwater dolphin, human, flying fox and different species of fish (Channa striata, Oncorhynchus mykiss and Lates calcarifer). As there is no database available for S. iniae in the MLST database yet, only limited isolates from the global collection that can be analysed with the Australian isolates. The present study found that MLST results are less discriminative when compared to PFGE, but were very useful in pinpointing origin to a particular country, perhaps indicating little transfer of isolates between nations. MLST grouped together Australian, Thailand (QMA0187 and QMA0190), human isolates from Canada and USA regardless the geographic origin (QMA0130, QMA0133 and QMA0137) and also fish isolate from Canada (QMA0139). The similarity of the human isolates with the Australian isolates supporting PFGE results, which had a similar SmaI PFGE profiles to many of the fish isolates from Australia. However, MLST managed to distinguish isolates QMA0140 (dolphin/ USA), QMA0141 (dolphin/ USA), QMA0136 (human/ USA) and other international isolates from Israel (QMA0186 and QMA0188), Reunion Island (QMA0189). Despite the degree of heterogeneity in other methods used (serotyping, PFGE), MLST method showed a high homogeneity amongst S. iniae from Australia, perhaps reflecting the slow evolution of these genes and no accidental import of isolates. During the 12 years of isolates covered by our strain collection, there would appear to have been no evolution of these highly conserved genes within Australia. The variation in serotype within a single Sequence Type showed that there may be frequent horizontal gene transfer, or more rapid evolution of genes involved in synthesis and transport of capsular polysaccharide or other surface features. Moreover, the PFGE results indicate that there is more genetic plasticity in amongst the genome of S. iniae than indicated by then MLST. In order to gain a more discriminative epidemiological perspective of S. iniae, the results presented in this thesis suggest combination of different typing methods such as PFGE and MLST, with the latter providing an accurate means of determining nation of origin of strains (and therefore of great potential for biosecurity purposes) whilst PFGE may provide better discrimination of movement of local isolates within Australia.

Neste trabalho são apresentados resultados relacionados a estudos sobre o comportamento eletroquímico do lactato e do peróxido de hidrogênio em diversos eletrodos em meio de diferentes pHs utilizando voltametria cíclica. Também foi investigado o comportamento eletroquímico do peróxido de hidrogênio em eletrodos modificados com filmes de hexacianoferratos utilizando eletrodos de platina e carbono vítreo. A vantagem do uso do CTAB na modificação da superfície de carbono vítreo com Azul da Prússia foi confirmada no que se refere à melhora da sensibilidade e da estabilidade das medidas. Numa etapa posterior desenvolveu-se um biossensor para a determinação de lactato pelo monitoramento de peróxido de hidrogênio produzido na reação catalisada de lactato com oxigênio na presença da enzima lactato oxidase. Nessa etapa, o trabalho consistiu em imobilizar a enzima lactato oxidase na superfície do eletrodo, previamente, modificado com Azul da Prússia, com o auxílio de Nafion®. Após a construção do biossensor e a otimização das condições de análise (pH, quantidade de enzima, volume da amostra e vazão) para obtenção de maior sinal analítico no desenvolvimento do método para a determinação de lactato por análise em fluxo, averiguou-se a repetibilidade das medidas (Clactato = 0,28 mmol L-1) obtendo-se um desvio padrão de 2,2% para 18 repetições. A freqüência analítica foi estimada em 160 injeções por hora com limite de detecção de 0,84 µmol L-1 e linearidade até 0,28 mmol L-1 de lactato. O biossensor foi aplicado na quantificação de lactato em amostras alimentares (cervejas alcoólicas e não alcoólicas) e biológicas (sangue liofilizado e recém coletado). Por fim, realizaram-se estudos envolvendo a variação da concentração de lactato sanguíneo em função da intensidade de atividade esportiva. Os resultados obtidos pelo método proposto foram comparados com aqueles oriundos do uso de método de referência. / Results on the investigation of the electrochemical behavior of lactate and hydrogen peroxide at various electrodic surfaces at different pHs using cyclic voltammetry are presented. Experiments were also carried out with platinum and glassy carbon electrodes modified with hexacyanoferrate films. The advantage of using CTAB in the electrodeposition step of Prussian Blue films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of measurements. The immobilization of lactate oxidase onto glassy carbon electrodes modified with a Prussian Blue layer using Nafion® was performed to fabricate a biosensor for lactate. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 mmol L-1 lactate with a limit of detection of 0.84 µmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The analytical frequency was calculated as 160 injections h-1. The usefulness of the method was demonstrated by determining lactate in beer (alcoholic and nonalcoholic beers) and blood samples (lyophilized and freshly collected). Finally, the influence of physical exercise on the blood lactate level was studied. Results obtained by using the proposed amperometric detector compared well with the reference method.

Les biocapteurs sont des moyens d'analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d'outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d'un pouvoir de reconnaissance pour un analyte ou un groupe d'analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de trois biocapteurs utilisant des enzymes de la famille des oxydo-réductases (alcool oxydase, lactate oxydase et formaldéhyde déshydrogénase) pour la détection d'analytes d'intérêt dans le domaine de l'agroalimentaire ou de l'environnement (alcools, lactate et formaldéhyde). Les trois enzymes ont été immobilisées sur des microélectrodes interdigitées en or en vue d'une détection conductimétrique. Nous avons cherché à mieux comprendre le fonctionnement des biocapteurs et optimisé le procédé d'immobilisation des enzymes ainsi que différents paramètres de fabrication ou de mesure afin de maximiser les performances analytiques des outils développés (sélectivité, limite de détection, reproductibilité, stabilité). Nous avons également démontré les potentialités d'application des deux biocapteurs conductimétriques à base de lactate oxydase et de formaldéhyde déshydrogénase pour la détection du lactate dans les produits laitiers et du formaldéhyde dans les eaux / Biosensors are rapid, selective and inexpensive devices that combine a biological recognition element, the so-called bioreceptor (e.g. enzymes, antibodies, DNA or microorganisms) to a physical transducer (e.g. electrochemical, optical, thermal or piezoelectrical). They can be used to detect one specific analyte or one family of analytes for a wide range of applications (e.g. environment, food, health). In this work, we developed biosensors based on three oxidoreductases (alcohol oxidase, lactate oxidase and formaldehyde dehydrogenase) for primary alcohols, lactate and formaldehyde analysis. The three enzymes were immobilized on interdigitated gold microelectrodes in view of conductometric detection. We tried to achieve a better understanding of biosensors processing and optimized enzyme immobilization as well as several fabrication or operational parameters to maximize analytical performances (selectivity, detection limit, reproducibility and stability). We also demonstrated the applicability of both conductometric biosensors based on lactate oxidase and formaldehyde dehydrogenase for the detection of lactate in dairy products and formaldehyde in waters