Toxoplasmosis in Indo-Pacific humpbacked dolphins (Sousa chinensis) from Queensland

Objective: To describe the clinical signs, gross pathology, serology, bacteriology, histopathology, electron microscopy and immunohistochemistry findings associated with toxoplasmosis in four Indo-Pacific humpbacked dolphins (Sousa chinensis) that stranded in Queensland in 2000 and 2001. Design: Clinical assessment, gross necropsy, and laboratory examinations. Procedure: Necropsies were performed on four S chinensis to determine cause of death. Laboratory tests including serology, bacteriology, histopathology and transmission electron microscopy were done on the four dolphins. lmmunohistochemistry was done on the brain, heart, liver, lung, spleen and adrenal gland from various dolphins to detect Toxoplasma gondii antigens. Results: Necropsies showed all of four S chinensis that stranded in Queensland in 2000 and 2001 had evidence of predatory shark attack and three were extremely emaciated. Histopathological examinations showed all four dolphins had toxoplasmosis with tissue cysts resembling T gondii in the brain. Tachyzoite stages of T gondii were detected in the lungs, heart, liver, spleen and adrenal gland, variously of all four dolphins. Electron microscopy studies and immunohistochemistry confirmed the tissues cysts were those of Tgondii. All four dolphins also had intercurrent disease including pneumonia, three had peritonitis and one had pancreatitis. Conclusion: Four S chinensis necropsied in Queensland in 2000 and 2001 were found to be infected with toxoplasmosis. It is uncertain how these dolphins became infected and further studies are needed to determine how S chinensis acquire toxoplasmosis. All four dolphins stranded after periods of heavy rainfall, and coastal freshwater runoff may be a risk factor for T gondii infection in S chinensis. This disease should be of concern to wildlife managers since S chinensis is a rare species and its numbers appear to be declining.

Development and validation of an ELISA for detecting antibodies to Eimeria tenella in chickens

The aim of this study was to develop and validate an ELISA for detecting chicken antibodies to Eimeria tenella. An initial comparison of merozoite and sporozoite antigen preparations revealed few differences in their ability to monitor the onset, kinetics and magnitude of the antibody response suggesting that both antigens would be equally useful for development of an ELISA. Furthermore the cross-reactivity of these antigens with sera from birds infected with chicken Eimeria species was similar. The merozoite antigen was selected for further evaluation because it was easier to prepare. Discrimination between sera from birds experimentally infected with E. tenella and birds maintained in an Eimeria-free isolation facility was excellent. In sera collected from free-range layers and commercial broilers there also appeared to be clear discrimination between infected and uninfected birds. The ELISA should prove useful for monitoring infectivity in vaccination programmes in layer and breeder flocks and for assessing the effectiveness of biosecurity measures in broiler flocks.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Floating row covers and pyriproxyfen help control silverleaf whitefly Bemisia tabaci (Gennadius) Biotype B (Homoptera: Aleyrodidae) in zucchini

In zucchini, the use of row covers until flowering and the insect growth regulator (IGR) pyriproxyfen are effective methods of reducing the number of insects, especially silverleaf whitefly (Bemisia tabaci (Gennadius) Biotype B), on plants. We compared floating row covers (FRCs) up until flowering with silverleaf whitefly (SLW) introduced (FRC + SLW), or not introduced (FRC-only), or with introduction of SLW in open plots (SLW-only), or with introduction of SLW in open plots with IGR (SLW + IGR). FRC increased temperature and humidity compared with the uncovered treatments. Average fruit weight was less (P < 0.01) for the FRC + SLW treatment compared with the other treatments and the percentage of marketable fruit was less for the FRC + SLW than for the other three treatments. This result indicates that the use of either row covers or IGR controls whiteflies, reduces fruit damage and increases the size, weight, and quality of fruit, and may also control other sap-sucking insects. However, if SLW are already present on plants, the use of FRC may reduce predation and favour build up of SLW. Thus, FRC and IGR, if used judiciously, may provide an effective alternative to broad-spectrum pesticides in small-scale cucurbit production.

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome b sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.

Sampling and management of Bemisia tabaci (Genn.) biotype B in Australian cotton

Data on seasonal population abundance of Bemisia tabaci biotype B (silverleaf whitefly (SLW)) in Australian cotton fields collected over four consecutive growing seasons (2002/2003-2005/2006) were used to develop and validate a multiple-threshold-based management and sampling plan. Non-linear growth trajectories estimated from the field sampling data were used as benchmarks to classify adult SLW field populations into six density-based management zones with associated control recommendations in the context of peak flowering and open boll crop growth stages. Control options based on application of insect growth regulators (IGRs) are recommended for high-density populations (>2 adults/leaf) whereas conventional (non-IGR) products are recommended for the control of low to moderate population densities. A computerised re-sampling program was used to develop and test a binomial sampling plan. Binomial models with thresholds of T=1, 2 and 3 adults/leaf were tested using the field abundance data. A binomial plan based on a tally threshold of T=2 adults/leaf and a minimum sample of 20 leaves at nodes 3, 4 or 5 below the terminal is recommended as the most parsimonious and practical sampling protocol for Australian cotton fields. A decision support guide with management zone boundaries expressed as binomial counts and control options appropriate for various SLW density situations is presented. Appropriate use of chemical insecticides and tactics for successful field control of whiteflies are discussed.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Pyriproxyfen controls silverleaf whitefly, Bemisia tabaci (Gennadius), biotype B (Homoptera: Aleyrodidae) (SLW) better than buprofezin in bitter melons Momordica charantia L. (Cucurbitaceae)

To improve compatibility between chemical and biological controls, the use of selective insecticides such as insect growth regulators (IGRs) is crucial. In cucurbits, the use of pyriproxyfen (an IGR) has been shown by others to be an effective method of reducing the number of sap-sucking insects, especially silverleaf whitefly, Bemisia tabaci (Gennadius) Biotype B (SLW). Therefore, we compared pyriproxyfen and buprofezin (an IGR) with that of no treatment (control) in a bitter melon crop for the control of populations of SLW and for their effects on fruit production. Pyriproxyfen controlled SLW and tended to have heavier fruits than the control treatment and reduced the abundance of nymphs and exuvia. Buprofezin showed no evidence in controlling SLW compared with the pyriproxyfen and control treatments. Neither pyriproxyfen nor buprofezin had any effect on the number of harvested fruit or overall fruit yield, but the average weight per fruit was higher than the control treatment. Pyriproxyfen was effective in controlling whitefly populations in bitter melons, and both pyriproxyfen and buprofezin may have the potential to increase yield. Their longer-term use may increase predation by natural enemies as they are species-specific and could favour build up of natural enemies of SLW. Thus, the judicious use of pyriproxyfen may provide an effective alternative to broad-spectrum insecticides in small-scale cucurbit production.

New efforts at biological control of Mikania micrantha H.B.K. (Asteraceae) in Papua New Guinea and Fiji.

New efforts at biological control of Miconia calvescens (Melastomataceae) is a serious invader in the tropical Pacific, including the Hawaiian and Tahitian Islands, and currently poses a major threat to native biodiversity in the Wet Tropics of Australia. The species is fleshy-fruited, small-seeded and shade tolerant, and thus has the potential to be dispersed widely and recruit in relatively intact rainforest habitats, displacing native species. Understanding and predicting the rate of spread is critical for the design and implementation of effective management actions. We used an individual-based model incorporating a dispersal function derived from dispersal curves for similar berry-fruited native species, and life-history parameters of fecundity and mortality to predict the spatial structure of a Miconia population after a 30 year time period. We compared the modelled population spatial structure to that of an actual infestation in the rainforests of north Queensland. Our goal was to assess how well the model predicts actual dispersion and to identify potential barriers and conduits to seed movement and seedling establishment. The model overpredicts overall population size and the spatial extent of the actual infestation, predicting individuals to occur at a maximum 1,750 m from the source compared with the maximum distance of any detected individual in the actual infestation of 1,191 m. We identify several characteristic features of managed invasive populations that make comparisons between modelled outcomes and actual infestations difficult. Our results suggest that the model’s ability to predict both spatial structure and spread of the population will be improved by incorporating a spatially explicit element, with dispersal and recruitment probabilities that reflect the relative suitability of different parts of the landscape for these processes. Mikania micrantha H.B.K. (Asteraceae) in Papua New Guinea and Fiji.

Inter-seasonal population dynamics and pest status of Bemisia tabaci (Gennadius) biotype B in an Australian cropping system

Bemisia tabaci, biotype B, commonly known as the silverleaf whitefly (SLW) is an alien species that invaded Australia in the mid-90s. This paper reports on the invasion ecology of SLW and the factors that are likely to have contributed to the first outbreak of this major pest in an Australian cotton cropping system, population dynamics of SLW within whitefly-susceptible crop (cotton and cucurbit) and non-crop vegetation (sowthistle, Sonchus spp.) components of the cropping system were investigated over four consecutive growing seasons (September-June) 2001/02-2004/05 in the Emerald Irrigation Area (EIA) of Queensland, Australia. Based on fixed geo-referenced sampling sites, variation in spatial and temporal abundance of SLW within each system component was quantified to provide baseline data for the development of ecologically sustainable pest management strategies. Parasitism of large (3rd and 4th instars) SLW nymphs by native aphelinid wasps was quantified to determine the potential for natural control of SLW populations. Following the initial outbreak in 2001/02, SLW abundance declined and stabilised over the next three seasons. The population dynamics of SLW is characterised by inter-seasonal population cycling between the non-crop (weed) and cotton components of the EIA cropping system. Cotton was the largest sink for and source of SLW during the study period. Over-wintering populations dispersed from weed host plant sources to cotton in spring followed by a reverse dispersal in late summer and autumn to broad-leaved crops and weeds. A basic spatial source-sink analysis showed that SLW adult and nymph densities were higher in cotton fields that were closer to over-wintering weed sources throughout spring than in fields that were further away. Cucurbit fields were not significant sources of SLW and did not appear to contribute significantly to the regional population dynamics of the pest. Substantial parasitism of nymphal stages throughout the study period indicates that native parasitoid species and other natural enemies are important sources of SLW mortality in Australian cotton production systems. Weather conditions and use of broad-spectrum insecticides for pest control are implicated in the initial outbreak and on-going pest status of SLW in the region.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3+, CD4+, CD8+ and [gamma][delta] T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for [gamma][delta] T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Evaluation of wood characteristics of tropical post-mid rotation plantation Eucalyptus cloeziana and E. pellita: Part (b) Accelerated seasoning of sawn timber.

Drying trials were conducted using two species of plantation grown eucalypt timbers: 19-year-old Eucalyptus cloeziana (Gympie messmate) and 15-year-old Eucalyptus pellita (red mahogany). The objective of this study was to gain an understanding of the drying potential of young plantation grown material using accelerated seasoning methods, a process expected to be critcal to the success of plantation hardwood products entering value added markets. The findings are encouraging, indicating that both species can be dried using conventional drying techniques much faster than industry is currently achieving when drying native forest timber. The results suggest that there is a definite drying time advantatge in vacuum drying over conventional methods for 19-year-old E. cloeziana. The findings have shown that through careful schedule manipulation and adjustment, the grade quality can be optimised to suit the desired expectation. As this study was limited to only a small number of trials, time and quality improvements are expected to be realised for both conventional and vacuum drying methods as more research is conducted.

Effectiveness of Various Artificial Larval Diets for Rearing Bactrocera passiflorae (Froggatt) and B. xanthodes (Broun) in the Laboratory in Fiji

Laboratory colonies of Bactrocera passiflorae (Froggatt) and B. xanthodes (Broun) were established at Koronivia Research Station, Fiji in 1991. Laboratory rearing of the two economically important species was a prerequisite to studies conducted on protein bait spray and quarantine treatment development. To increase the production of laboratory reared fruit flies for this research and also to have a substitute larval diet available, replicated comparisons of the effectiveness of larval diets were carried out using B. passiflorae and B. xanthodes. The diets compared were pawpaw/bagasse, dehydrated carrot and diets used for culturing Mediterranean fruit fly (Ceratitis capitata Wiedemann), Oriental fruit fly (B. dorsalis Hendel), melon fly (B. cucurbitae Coquillett) and B. latifrons (Hendel), pawpaw diet and breadfruit diet. B. passiflorae and B. xanthodes eggs seeded onto the various diets were allowed to develop into larvae, pupae and adults. The percentage egg hatch, number of pupae recovered, percentage pupal mortality, weight of 100 pupae, number of adults and percentage eclosion were used to determine the effectiveness of the diets. Results showed that pawpaw/bagasse and dehydrated carrot diets performed favorably for both species. The pawpaw diet currently used as standard larval diets for both species is the most readily available and easiest to use. Breadfruit diet was tested on B. xanthodes only and showed that it was a suitable substitute for the pawpaw-based diets. Other larval diets, cassava/pawpaw and banana diets, that have been developed and used in the South Pacific areas are also discussed in this paper. When pawpaw or breadfruit are not available, dehydrated carrot diet may be substituted for fruit-based larval diets.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value = 0.66) that increased to very good (k-value = 0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P < 0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing. (C) 2013 Elsevier B.V. All rights reserved.