Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Some mounted ill. / Bibliography: leaves 156-186 / ix, 186, 43 leaves, 35 leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, Waite Agricultural Research Institute, 1984

Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

Conservation biology is primarily concerned with the amelioration of species decline. The Yellow-footed Rock-wallaby (Petrogale xanthopus xanthopus) is a medium-sized Macropod that inhabits the semiarid rangelands of South Australia and New South Wales. Its conservation status is Vulnerable C2a(i). In this study, population modelling, spatially explicit habitat modelling and Population Viability Analysis (PVA) have been used to better understand the factors that affect the abundance and distribution of the P. x. xanthopus in South Australia. The processes that drive the population dynamics of a species operate at different scales. As such this research involves a collection of several inter-related and scale-specific empirical studies that provide insights about the population dynamics of P. x. xanthopus. Each of these studies captures environmental, demographic and behavioural process acting on the population at different scales. These include the analysis of relative abundance data derived from an aerial census, mark recapture sampling of demographic parameters in relation to rainfall patterns and a collection of habitat models derived at different scales using presence-absence data. Spatially explicit PVAs enable the population dynamics of a species to be modelled in space and time. Using these data, a PVA is conducted to explore and rank the importance of the factors that threaten this species and help guide their future monitoring and management. Movement is also a key issue when considering problems such as isolation and inbreeding. Given that little is known about the dispersal behaviour of this species, a range of different dispersal behaviours are also simulated in the PVA using random and non-random mating algorithms, to estimate the potential for inbreeding. / thesis (PhD)--University of South Australia, 2004.

Yellow head virus (YHV) is highly pathogenic and was identified as the cause of mass mortalities associated with yellow head disease (YHD) that first appeared in Penaeus monodon farmed in Thailand in 1990. By 1992-1993, YHD was widespread throughout the Thai shrimp farming industry, causing losses estimated at ~US$70 million per annum. By the mid 1990s, gross signs consistent with YHD were also being reported in P. monodon farmed in many regions of the Indo-Pacific. Due to its high pathogenicity and economic impact, YHV has been listed as a notifiable pathogen by the OIE and the control of YHD remains a significant concern. At the outset of this study, two genotypic variants of YHV (genotype 1) had been detected in P. monodon in Australia (gill-associated virus, GAV, genotype 2) and Vietnam (genotype 3), suggesting that more variants might exist in other regions. The aim of this study was, therefore, to test the hypothesis that genotypic variants existed in P. monodon from other locations, and if so, to determine their genetic relationships to the three known genotypes. The study also aimed to improve existing PCR diagnostic protocols to accommodate the detection of all genotypes in the YHV complex. Fifty-seven field isolates of YH-complex viruses were detected by RT-PCR in tissues of P. monodon sampled from nine Indo-Pacific countries. Phylogenetic relationships determined for these isolates using a 671 nucleotide (nt) C-terminal region of the ORF1b gene identified 46 isolates that clustered with the three know genotypes and 11 isolates that clustered in at least three distinct new genotypes. All isolates other than genotype 1 (YHV) were detected in tissues of healthy shrimp. Genotype 4 isolates were detected only in shrimp from India and were slightly less distantly related at the nucleotide level to genotype 5 (85.2% identify) than the other genotypes (80.3%-82.3%). Genotype 6 isolates were only detected in shrimp from Mozambique and were least divergent (3.5%) from genotype 2. One each of three genotype 5 isolates was detected in shrimp from Malaysia, Thailand and the Philippines. The genotype 5 isolate from the Philippines was, however, 6.7% and 7% divergent from the other two isolates, respectively. This level of divergence was greater than found between genotypes 2 and 6 and was similar to that found between isolates of genotype 2 and genotype 3 (~6.7%). This suggests that the Philippine genotype 5 isolate might ultimately be considered as the founding member of a seventh genotype. Genotype 5 isolates were slightly more closely related to genotype 4 (~85.2% identity) than the other genotypes (83.4%-84.8% identity). Genotype 1 (YHV) isolates were only detected in Thai shrimp affected by YHD. Genotype 2 isolates were detected in Australian shrimp as well as shrimp from Vietnam and Thailand. Genotype 3 had the broadest geographic range, being detected in four countries in Southeast Asia. The finding of single genotypes in Australia (genotype 2), India (genotype 4) and Mozambique (genotype 6) supports the hypothesis that they have evolved independently in geographically-isolated populations of P. monodon. The detection of multiple genotypes in Vietnam (genotypes 2 and 3), Malaysia (genotypes 2, 3 and 5) and Thailand (genotypes 1, 2, 3 and 5) suggests that these genotypes have been disseminated by movements of infected P. monodon and the trade in live broodstock used for aquaculture. A ~1.3 kb amplicon at the 5’-terminal region of the ORF3 gene was sequenced for 28 field isolates to examine phylogenetic relationships to assess whether there is evidence of recombination between genotypes. The region, corresponding to N-terminus of gp116 envelope glycoprotein, displayed more sequence variation than the ORF1b amplicon. All isolates of the virulent genotype 1 (YHV) possessed a unique sequence (TILAGIPEKE/D) at the N terminus of gp116 adjacent to the site of endo-proteolysis that cleaves gp116 from the ORF3 polyprotein. In some genotype 1 isolates this unique sequence was followed by a 54 aa deletion that was also not present in other genotypes. The potential role of this unique sequence as a virulence determinant for YHV requires further investigation. Phylogenetic relationships deduced using the ORF3 amplicon sequences were similar to those deduced using the ORF1b amplicon sequence except that genotype 4 was more closely related to genotype 2 than was genotype 3. However, only 18 of the 28 isolates included in the analysis of both ORF1b and ORF3 amplicons clustered in consistent lineages and were assigned as the same genotypes. Inconsistent phylogenies were observed for ten isolates of which six clustered as genotype 3 in ORF1b and as genotype 2 in ORF3, two isolates clustered as genotype 3 in ORF1b and as genotype 5 in ORF3, one isolate clustered as genotype 5 in ORF1b and as genotype 2 in ORF3, and one isolate clustered as genotype 5 in ORF1b and as genotype 3 in ORF3. Discrepancies in genotype assignments were only observed to involve permutations of genotypes 2, 3 and 5 and involved isolates from healthy shrimp originating from Southeast Asia. Sequence analysis of the ~3.2 kb region spanned by the ORF1b and ORF3 amplicons of three putative recombinant viruses VNM-02-H258 (genotype 3/5), IDN-04-H10 (genotype 3/2) and PHL-03-H8 (genotype 5/3) indicated that recombination had occurred at a position just upstream of the ORF1b gene 3’-terminus. These data provide the first evidence of genetic recombination for any shrimp virus. The high prevalence of recombinants amongst isolates from Southeast Asia has significant implications for diversification, disease emergence and assignment of genotypes for YH-complex viruses. The region of the genome from the poly[A] tail to the 3’-end of the ORF1b gene (containing all structural protein genes) was sequenced for representative isolates of genotypes 3 and 4. The analysis was conducted to determine whether the evolutionary divergence in the structural protein genes differed significantly from the replicase (ORF1b) gene and to identify conserved motifs likely to be important for protein function and the regulation of RNA transcription and replication. The sequence of the near 3’-terminal genome region of a genotype 5 isolate was also determined to examine whether it possessed an ORF4 gene like genotype 2 or whether it was truncated as in genotypes 1, 3 and 4. Comparisons of the intergenic regions (IGR) upstream of ORF2 and ORF3 identified a conserved sequence 5’-GUCAAUUACACxxAxxUU-3’ surrounding the central adenosine residue corresponding to the 5’-terminus of the sub-genomic (sg)mRNAs that is likely to represent the consensus motif used as a transcription regulatory sequence (TRS). A sequence upstream of ORF4 possessed limited homology to the predicted consensus TRS but A>G/U substitutions (genotypes 2, 3, 4 and 5) or a point deletion (genotype 1) occurred at the central critical adenosine residue. It is possible that these mutations explain why a sgmRNA is not transcribed in abundance to allow translation of an ORF4 protein, and why the apparently redundant ORF4 gene has accumulated nucleotide deletions or insertions interrupting its reading in all genotypes except genotype 2. The 3’-terminal genome sequence of genotypes 1, 2, 3 and 4 downstream of the putative ORF4 gene region was extremely highly conserved and was predicted to form a stable hairpin-loop RNA secondary structure with four bulges. Where nucleotide variations occurred in a genotype, other compensatory changes maintained base-pairing and stability of the structure, suggesting that this region is likely to be important for polymerase recognition of the (+) genomic RNA for transcription of (-) genomic RNA. Conventional and real-time PCR tests for the detection of all genotypes in the YH complex were developed by identifying highly conserved sequences amongst the 57 virus isolates at which primers could be targeted. In the consensus RT-nested PCR, PCR (358 bp) and nested PCR (147 bp) amplicon lengths were kept short to accommodate degraded RNA and pools of two primers were used rather than a single degenerate primer to accommodate all genotypes whist minimizing levels of degeneracy. The consensus real-time PCR used SYBR-Green chemistry and amplified a 147 bp product using single degenerate primers targeted to the same sites as the nested PCR primer pools. Each PCR method detected the RNA of representatives of all six genotypes. The RT-nested PCR was extremely sensitive, detecting down to a single copy of a GAV synthetic RNA. Phylogenetic analysis using the 95 nt sequence bounded by the nested PCR primers generated genotype associations similar to those generated using the 671 nt sequence, allowing the assignment of genotypes from the amplified products. The consensus RT-nested PCR test has been included in the 5th Edition of the OIE Manual of Diagnostic Tests for Aquatic Animals (2006). The consensus real-time PCR was slightly less sensitive than the RT-nested PCR, detecting down to ~125 copies of the GAV synthetic RNA. However, the test generated products with the expected Tm (77.5ºC) with isolates of the six genotypes and showed a linear relationship between input RNA and Ct value up to 109 RNA copies. Thus, due to its ability to accurately quantify and compare viral RNA loads in clinical samples, the test could be used to define the infection status of shrimp in relation to threshold levels associated with disease.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 274-319).

Thesis (M.S.)--Naval Postgraduate School, 1994. / "September 1994." Includes bibliographical references (p. 77-78).

Thesis (M.S.)--Wildlife and Fisheries Sciences Dept., South Dakota State University, 2009. / Includes bibliographical references (leaves 80-102). Also available via the World Wide Web.

Thesis--Universidad de Sevilla. / "Apéndice demografico": p. 134-180. Includes bibliographical references (p. 132-134).