Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis

The aim of this study was to compare the use of indirect haemagglutination (IHA) and gel diffusion (GD) tests for serotyping Haemophilus parasuis by the Kielstein-Rapp-Gabrielson scheme. All 15 serovar reference strains, 72 Australian field isolates, nine Chinese field isolates, and seven isolates from seven experimentally infected pigs were evaluated with both tests. With the IHA test, 14 of the 15 reference strains were correctly serotyped – with serovar 10 failing to give a titre with serovar 10 antiserum. In the GD test, 13 reference strains were correctly serotyped – with antigen from serovars 7 and 8 failing to react with any antiserum. The IHA methodology serotyped a total of 45 of 81 field isolates while the GD methodology serotyped a total of 48 isolates. For 29 isolates, the GD and IHA methods gave discordant results. It was concluded that the IHA is a good additional test for the serotyping of H. parasuis by the KRG scheme if the GD methodology fails to provide a result or shows unusual cross-reactions.

New annual and short-lived perennial pasture legumes for Australian agriculture--15 years of revolution

Fifteen years ago subterranean clover (Trifolium subterraneum) and annual medics (Medicago spp.) dominated annual pasture legume sowings in southern Australia, while limited pasture legume options existed for cropping areas of subtropical Australia. Since then a number of sustainability and economic challenges to existing farming systems have emerged, exposing shortcomings in these species and the lack of legume biodiversity. Public breeding institutions have responded to these challenges by developing 58 new annual and short-lived perennial pasture legumes with adaptation to both existing and new farming systems. This has involved commercialisation of new species and overcoming deficiencies in traditional species. Traits incorporated in legumes of Mediterranean Basin origin for the Mediterranean, temperate and southern subtropical climates of Australia include deeper root systems, protection from false breaks (germination-inducing rainfall events followed by death from drought), a range of hardseed levels, acid-soil tolerant root nodule symbioses, tolerance to pests and diseases and provision of lower cost seed through ease of seed harvesting and processing. Ten new species, French serradella (Ornithopus sativus), biserrula (Biserrula pelecinus), sulla (Hedysarum coronarium), gland (Trifolium glanduliferum), arrowleaf (Trifolium vesiculosum), eastern star (Trifolium dasyurum) and crimson (Trifolium incarnatum) clovers and sphere (Medicago sphaerocarpos), button (Medicago orbicularis) and hybrid disc (Medicago tornata x Medicago littoralis) medics have been commercialised. Improved cultivars have also been developed of subterranean (T. subterraneum), balansa (Trifolium michelianum), rose (Trifolium hirtum), Persian (Trifolium resupinatum) and purple (Trifolium purpureum) clovers, burr (Medicago polymorpha), strand (M. littoralis), snail (Medicago scutellata) and barrel (Medicago truncatula) medics and yellow serradella (Ornithopus compressus). New tropical legumes for pasture phases in subtropical cropping areas include butterfly pea (Clitoria ternatea), burgundy bean (Macroptilium bracteatum) and perennial lablab (Lablab purpureus). Other species and cultivars of Mediterranean species are likely to be released soon. The contributions of genetic resources, rhizobiology, pasture ecology and agronomy, plant pathology, entomology, plant chemistry and animal science have been paramount to this success. A farmer survey in Western Australia has shown widespread adoption of the new pasture legumes, while adoption of new tropical legumes has also been high in cropping areas of the subtropics. This trend is likely to increase due to the increasing cost of inorganic nitrogen, the need to combat herbicide-resistant crop weeds and improved livestock prices. Mixtures of these legumes allows for more robust pastures buffered against variable seasons, soils, pests, diseases and management decisions. This paper discusses development of the new pasture legumes, their potential use and deficiencies in the current suite. 'Ground–breaking Stuff’- Proceedings of the 13th Australian Society of Agronomy Conference, 10-14 September 2006, Perth, Western Australia.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

Characterization of north-eastern Australian environments using APSIM for increasing rainfed maize production

Recurring water stresses are a major risk factor for rainfed maize cropping across the highly diverse agro-ecological environments of Queensland (Qld) and northern New South Wales (NNSW). Enhanced understanding of such agro-ecological diversity is necessary to more consistently sample target production environments for testing and targeting release of improved germplasm, and to improve the efficiency of the maize pre-breeding and breeding programs of Qld and New South Wales. Here, we used the Agricultural Production Systems Simulator (APSIM) – a well validated maize crop model to characterize the key distinctive water stress patterns and risk to production across the main maize growing regions of Qld and NNSW located between 15.8° and 31.5°S, and 144.5° and 151.8°E. APSIM was configured to simulate daily water supply demand ratios (SDRs) around anthesis as an indicator of the degree of water stress, and the final grain yield. Simulations were performed using daily climatic records during the period between 1890 and 2010 for 32 sites-soils in the target production regions. The runs were made assuming adequate nitrogen supply for mid-season maize hybrid Pioneer 3153. Hierarchical complete linkage analyses of the simulated yield resulted in five major clusters showing distinct probability distribution of the expected yields and geographic patterns. The drought stress patterns and their frequencies using SDRs were quantified using multivariate statistical methods. The identified stress patterns included no stress, mid-season (flowering) stress, and three terminal stresses differing in terms of severity. The combined frequency of flowering and terminal stresses was highest (82.9%), mainly in sites-soils combinations in the west of Qld and NNSW. Yield variability across the different sites-soils was significantly related to the variability in frequencies of water stresses. Frequencies of water stresses within each yield cluster tended to be similar, but different across clusters. Sites-soils falling within each yield cluster therefore could be treated as distinct maize production environments for testing and targeting newly developed maize cultivars and hybrids for adaptation to water stress patterns most common to those environments.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.