Coliforms in processed mango: Significance and control

The aims of this investigation were to enumerate coliforms in fresh mangoes, puree, cheeks, and cheeks-in-puree in order to determine the source of these organisms in the processed products, to determine methods for their control, and to identify coliforms isolated from cheeks-in-puree to determine whether they have any public health significance. Product from four processors was tested on two occasions. The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 × 103 and 5.4 × 104 cfu/g. Pasteurisation reduced the coliform count of raw puree to < 5 cfu/g. Forty-seven percent of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as Klebsiella pneumoniae using the ATB 32E Identification System. Klebsiella strains were tested for growth at 10 °C, faecal coliform response, and fermentation of -melizitose, to differentiate the three phenotypically similar strains, K. pneumoniae, K. terrigena and K planticola. Results indicated that 41% of K. pneumoniae isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern for these tests and were designated ‘psychrotrophic’ K. pneumoniae. Klebsiella pneumoniae counts of between 2.1 × 103 and 4.9 × 104 cfu/g were predicted to occur in the retail packs of mango cheeks-in-puree produced by the processors who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurisation of puree, should be taken in order to inactivate it

On Systems Thinking, Systems Biology, and the in Silico Plant

The recent summary report of a Department of Energy Workshop on Plant Systems Biology (P.V. Minorsky [2003] Plant Physiol 132: 404-409) offered a welcomed advocacy for systems analysis as essential in understanding plant development, growth, and production. The goal of the Workshop was to consider methods for relating the results of molecular research to real-world challenges in plant production for increased food supplies, alternative energy sources, and environmental improvement. The rather surprising feature of this report, however, was that the Workshop largely overlooked the rich history of plant systems analysis extending over nearly 40 years (Sinclair and Seligman, 1996) that has considered exactly those challenges targeted by the Workshop. Past systems research has explored and incorporated biochemical and physiological knowledge into plant simulation models from a number of perspectives. The research has resulted in considerable understanding and insight about how to simulate plant systems and the relative contribution of various factors in influencing plant production. These past activities have contributed directly to research focused on solving the problems of increasing biomass production and crop yields. These modeling approaches are also now providing an avenue to enhance integration of molecular genetic technologies in plant improvement (Hammer et al., 2002).

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Microbiological status of piggery effluent from 13 piggeries in the south east Queensland region of Australia

Aims: To assist in the development of safe piggery effluent re-use guidelines by determining the level of selected pathogens and indicator organisms in the effluent ponds of 13 south-east Queensland piggeries. Methods and Results: The numbers of thermotolerant coliforms, Campylobacter jejuni/coli, Erysipelothrix rhusiopathiae, Escherichia coli, Salmonella and rotavirus were determined in 29 samples derived from the 13 piggeries. The study demonstrated that the 13 final effluent ponds contained an average of 1Æ2 · 105 colony-forming units (CFU) 100 ml)1 of thermotolerant coliforms and 1Æ03 · 105 CFU 100 ml)1 of E. coli. The Campylobacter level varied from none detectable (two of 13 piggeries) to a maximum of 930 most probable number (MPN) 100 ml)1 (two of 13 piggeries). Salmonella was detected in the final ponds of only four of the 13 piggeries and then only at a low level (highest level being 51 MPN 100 ml)1). No rotavirus and no Erysip. rhusiopathiae were detected. The average log10 reductions across the ponding systems to the final irrigation pond were 1Æ77 for thermotolerant coliforms, 1Æ71 for E. coli and 1Æ04 for Campylobacter. Conclusions: This study has provided a baseline knowledge on the levels of indicator organisms and selected pathogens in piggery effluent. Significance and Impact of the Study: The knowledge gained in this study will assist in the development of guidelines to ensure the safe and sustainable re-use of piggery effluent.

Candidatus Phytoplasma australiense is associated with pumpkin yellow leaf curl disease in Queensland, Western Australia and the Northern Territory

Pumpkin plants (Cucurbita maxima and C. moschata) with pumpkin yellow leaf curl (PYLC) disease were observed at production fields in Queensland, Western Australia and the Northern Territory. Diseased samples were positive for a phytoplasma indistinguishable from Candidatus Phytoplasma australiense, the phytoplasma associated with papaya dieback and strawberry lethal yellows. This is the first time Candidatus Phytoplasma australiense has been detected in pumpkin.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction.

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

Aims: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. Methods and Results: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 × 105 to 1.1 × 108 MPN 100 ml-1 and in freshly irrigated soils from 9.5 × 102 to 2.8 × 104 MPN g-1 in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. Conclusions: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. Significance and Impact of the Study: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.

The use of F-specific coliphages to assess effluent treatment and reuse schemes

This study reports on the use of naturally occurring F-specific coliphages, as well as spiked MS-2 phage, to evaluate a land-based effluent treatment/reuse system and an effluent irrigation scheme. Both the natural phages and the spiked MS-2 phage indicated that the effluent treatment/reuse system (FILTER - Filtration and Irrigated cropping for Land Treatment and Effluent Reuse) achieved a reduction in phage levels over the treatment system by one to two log10. FILTER reduced natural F-specific phage numbers from around 103 to below 102 100-ml-1 and the spiked phage from 105 to around 104 100-ml-1 (incoming compared with outgoing water). In the effluent irrigation scheme, phage spiked into the holding ponds dropped from 106 to 102 100-ml-1 after 168 h (with no detectable levels of natural F-specific phage being found prior to spiking). Only low levels of the spiked phage (102 gm-1) could be recovered from soil irrigated with phage-spiked effluent (at 106 phage 100 ml-1) or from fruits (around 102 phage per fruit) that had direct contact with soil which had been freshly irrigated with the same phage-spiked effluent.

Fungal colonization of sago starch in Papua New Guinea

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 × 103 to 8.3 × 107 cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 × 102 to 3.0 × 106 cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB?) labelled with 6-carboxy-fluorescein (FAM?) and VIC® for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.

Fishery biology and management of Protonibea diacanthus (Sciaenidae) aggregations in far Northern Cape York Peninsula waters

The sciaenid Protonibea diacanthus is a large, long-lived predatory fish of inshore northern Australian waters, which forms annual aggregations that are fished extensively by traditional (subsistence) and recreational fishers. There are now widespread concerns that the resource is being overexploited. Indigenous fishers of the Cape York Northern Peninsula Area (NPA) relate that large adult fish (up to 1500 mm total length (TL)) made up the bulk of the catch from the sciaenid aggregations until about 1994. In contrast, sexually mature P. diacanthus comprised only a small component (12 fish out of 270=4.4%) examined in a 1999–2000 sampling programme that was biased towards the largest individuals available. At 790 mm TL, the minimum size at first maturity for female P. diacanthus in this study is much smaller than the 920 mm TL reported previously in Queensland waters. Developing ovaries were observed in specimens sampled from sciaenid aggregations which formed in NPA waters between May and September 2000. However, no fish with ripe or spent gonads were found in the study, so the current timing and location of the spawning season for P. diacanthus in the region remain unknown. Food items observed in the analysis of the diet of P. diacanthus from the NPA included a variety of teleosts and invertebrates. The range of animal taxa represented in the prey items support the description of an ‘opportunistic predator’ attributed to the species. In our sampling, the stomach contents of fish caught during the time of the aggregation events did not differ from those observed at other times of the year. A total of 114 P. diacanthus were tagged and released at aggregation sites during the study period, and 3 fish (2.6%) were subsequently recaptured. The low rate of tag returns from the wild stock tagging programme, both in this study (2.6%) and from recreational fisher tag/release programmes for the sciaenid elsewhere in Queensland (6.5%), were not explained by tag loss nor mortality, given the high retention rate of tags and the zero mortality seen in tank trials. In response to the biological findings from this study, indigenous community councils of the NPA imposed a 2-year fishing moratorium for P. diacanthus. Surveys at aggregation sites in 2002 and 2003 established that much larger fish (mean size 103.5 cm TL) were again present on the grounds, albeit in very low numbers. These recent preliminary results highlight the critical need for continued monitoring and management of the P. diacanthus fishery in the NPA, if prospects for resource recovery are to be realised. The NPA initiative has provided a rare opportunity to negotiate a co-management strategy, based on scientific data and traditional knowledge, for the recovery of a cultural and economically significant fished resource.

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.

Spatial and temporal variation and effects of changes in management in discard rates from the commercial reef line fishery of the Great Barrier Reef, Australia

Discarding in commercially exploited fisheries has received considerable attention in the last decade, though only more recently in Australia. The Reef Line fishery (RLF) of the Great Barrier Reef (GBR) in Australia is a large-scale multi-sector, multi-species, highly regulated hook and line fishery with the potential for high levels of discarding. We used a range of data sources to estimate discard rates and discard quantities for the two main target groups of the RLF, the coral trout, Plectropomus spp, and the red throat emperor, Lethrinus miniatus, and investigated possible effects on discarding of recent changes in management of the fishery. Fleet-wide estimates of total annual quantities discarded from 1989 to 2003 were 292-622 t and 33-95 t for coral trout and red throat emperor, respectively. Hypothetical scenarios of high-grading after the introduction of a total allowable commercial catch for coral trout resulted in increases in discard quantities up to 3895 t, while no high-grading still meant 421 t were discarded. Increasing the minimum size limit of red throat emperor from 35 to 38 cm also increased discards to an estimated 103 t. We provide spatially and temporally explicit estimates of discarding for the two most important species in the GBR RLF of Australia to demonstrate the importance of accounting for regional variation in quantification of discarding. Effects of management changes on discarding are also highlighted. This study provides a template for exploring discarding levels for other species in the RLF and elsewhere.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Managing for rainfall variability: effect of grazing strategy on cattle production in a dry tropical savanna

Rainfall variability is a challenge to sustainable and pro. table cattle production in northern Australia. Strategies recommended to manage for rainfall variability, like light or variable stocking, are not widely adopted. This is due partly to the perception that sustainability and profitability are incompatible. A large, long-term grazing trial was initiated in 1997 in north Queensland, Australia, to test the effect of different grazing strategies on cattle production. These strategies are: (i) constant light stocking (LSR) at long-term carrying capacity (LTCC); (ii) constant heavy stocking (HSR) at twice LTCC; (iii) rotational wet-season spelling (R/Spell) at 1.5 LTCC; (iv) variable stocking (VAR), with stocking rates adjusted in May based on available pasture; and (v) a Southern Oscillation Index (SOI) variable strategy, with stocking rates adjusted in November, based on available pasture and SOI seasonal forecasts. Animal performance varied markedly over the 10 years for which data is presented, due to pronounced differences in rainfall and pasture availability. Nonetheless, lighter stocking at or about LTCC consistently gave the best individual liveweight gain (LWG), condition score and skeletal growth; mean LWG per annum was thus highest in the LSR (113 kg), intermediate in the R/Spell (104 kg) and lowest in the HSR(86 kg). MeanLWGwas 106 kg in the VAR and 103 kg in the SOI but, in all years, the relative performance of these strategies was dependent upon the stocking rate applied. After 2 years on the trial, steers from lightly stocked strategies were 60-100 kg heavier and received appreciable carcass price premiums at the meatworks compared to those under heavy stocking. In contrast, LWG per unit area was greatest at stocking rates of about twice LTCC; mean LWG/ha was thus greatest in the HSR (21 kg/ha), but this strategy required drought feeding in four of the 10 years and was unsustainable. Although LWG/ha was lower in the LSR (mean 14 kg/ha), or in strategies that reduced stocking rates in dry years like the VAR(mean 18 kg/ha) and SOI (mean 17 kg/ha), these strategies did not require drought feeding and appeared sustainable. The R/Spell strategy (mean 16 kg/ha) was compromised by an ill-timed fire, but also performed satisfactorily. The present results provide important evidence challenging the assumption that sustainable management in a variable environment is unprofitable. Further research is required to fully quantify the long-term effects of these strategies on land condition and profitability and to extrapolate the results to breeder performance at the property level.