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.

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.

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.

Virulence of the Nine Serovar Reference Strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.

A lack of predatory interaction between rumen ciliate protozoa and Shiga-toxin producing Escherichia coli

Aims: To investigate interactions between rumen protozoa and Shiga toxin-producing Escherichia coli (STEC) and to ascertain whether it is likely that rumen protozoa act as ruminant hosts for STEC. Methods and Results: The presence of stx genes in different microbial fractions recovered from cattle and sheep rumen contents and faeces was examined using PCR. In animals shedding faecal STEC, stx genes were not detected in the rumen bacterial or rumen protozoal fractions. Direct interactions between ruminal protozoa and STEC were investigated by in vitro co-incubation. Rumen protozoa did not appear to ingest STEC, a STEC lysogen or non-STEC E. coli populations when co-incubated. Conclusions: The ruminal environment is unlikely to be a preferred habitat for STEC. Bacterial grazing by rumen protozoa appears to have little, if any, effect on STEC populations. Significance and Impact of the Study: This study indicates that ruminal protozoa are unlikely to be a major factor in the survival of STEC in ruminants. They appear as neither a host that protects STEC from the ruminal environment nor a predator that might reduce STEC numbers.

Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus

We have characterised six Australian Cucumber mosaic virus (CMV) strains belonging to different subgroups, determined by the sequence of their complete RNA 3 and by their host range and the symptoms they cause on species in the Solanaceae, Cucurbitaceae and on sweet corn. These data allowed classification of strains into the known three CMV subgroups and identification of plant species able to differentiate the Australian strains by symptoms and host range. Western Australian strains 237 and Twa and Queensland strains 207 and 242 are closely related members of CMV subgroup IA, which cause similar severe symptoms on Nicotiana species. Strains 207 and 237 (subgroup IA) were the only strains tested which systemically infected sweet corn. Strain 243 caused the most severe symptoms of all strains on Nicotiana species, tomato and capsicum and appears to be the first confirmed subgroup IB strain reported in Australia. Based on pair-wise distance analysis and phylogeny of RNA 3, as well as mild disease symptoms on Nicotiana species, CMV 241 was assigned to subgroup II, as the previously described Q-CMV and LY-CMV.

Optimising indoor phosphine fumigation of paddy rice bag-stacks under sheeting for control of resistant insects

Three indoor, sheeted bag-stack fumigations of paddy rice using aluminium phosphide were undertaken in Guangdong Province, southern China. We measured the effect of two types of sheeting (polyvinylchloride [PVC] or polyethylene [PE]) and two types of floor sealing (clips or fixing into a slot with a rubber pipe) on phosphine concentration and retention. The aim was to test the feasibility of retaining fumigant at a sufficient concentration for long enough to control known resistant insect pests. Each stack was pressure tested and phosphine concentrations measured daily during the fumigation. Cages of test insects in culture medium, including resistant and susceptible strains, were placed inside each stack and could be observed through the clear sheeting. Highest concentrations for the longest period were obtained in a PVC-covered stack that included a ground sheet and wall sheets sealed to the floor with rubber pipes. A similar PVC-covered stack sealed to the floor with clips instead of pipe did not retain gas as efficiently and required re-dosing. A PE-covered stack, with no ground sheet but also with wall sheets sealed to the floor with pipe, produced an acceptable fumigation. Susceptible Rhyzopertha dominica were controlled in 2 days and the most resistant strain in 15 days. Resistant Cryptolestes ferrugineus survived until day 21. The paddy was still free of insect infestation 7 months later when the bag-stack was opened to mill the rice. Pressure half-lives correlated with gas concentration and retention. Sorption appeared to be a major limiting factor, reducing potential fumigant dosage by about 50%. The trials demonstrated the feasibility of sealing bag-stacks to a standard high enough to control all known resistant strains.

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.

Environmental regulation in the pork producing industry - bring on the research!

Over the last 20 years, environmental management in Queensland has moved from the policy backwaters of government to the front line of operations by way of regulatory enforcement, industry programs and incentives. When the new Queensland Environmental Protection Act 1994 (EPA) came into effect, the business of environmental management has become a central feature of urban and rural development activity. The concept of environmentally sustainable development (ESD), has given life to the precautionary principle as a way for planners and regulators to place relevant controls on development. The planning, development and operation of pig farming systems has been effected by the new regulatory framework. Ever more definitive standards and approval permits have emerged which endeavour to achieve ESD. With these modern planning instruments in place, rural industry sectors have become, quite legitimately, concerned about future opportunities for research and innovation. This paper asserts that the capacity to engage in research and to achieve innovation in the pork producing industry is not hindered by Queensland environmental regulation frameworks. However, in order for research and innovation to prosper within these frameworks, some protocols need to be followed by the industry. What is at stake is community confidence.

In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.

A novel method for screening bacterial isolates for their potential to inhibit the growth of ruminal methanogenic Archaea was developed using a modification of the soft agar overlay technique, formally used for the isolation of lytic bacteriophages. This method may be used in the specific, hydrogen-rich conditions required for the growth of ruminal methanogenic Archaea.

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants.

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin+ trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.

Controlled exposure as a management tool for Glasser's disease.

In this study, nasal swabs taken from multiparous sows at weaning time or from sick pigs displaying symptoms of Glasser's disease from farms in Australia [date not given] were cultured and analysed by polymerase chain reaction (PCR). Within each genotype detected on a farm, representative isolates were serotyped by gel diffusion (GD) testing or indirect haemagglutination (IHA) test. Isolates which did not react in any of the tests were regarded as non-typable and were termed serovar NT. Serovars 1, 5, 12, 13 and 14 were classified as highly pathogenic; serovars 2, 4 and 15 being moderately pathogenic; serovar 8 being slightly pathogenic and serovars 3, 6, 7, 9 and 11 being non-pathogenic. Sows were inoculated with the strain of Haemophilus parasuis (serovars 4, 6 and 9 from Farms 1, 2 and 4, respectively) used for controlled challenge 3 and 5 weeks before farrowing. Before farrowing the sows were divided into control and treatment groups. Five to seven days after birth, the piglets of the treatment group were challenged with a strain from the farm which had were used to vaccinate the sows. The effectiveness of the controlled exposure was evaluated by number of piglets displaying clinical signs possibly related to infection, number of antibiotic treatments and pig mortality. Nasal swabs of sick pigs were taken twice a week to find a correlation to infection. A subsample of pigs was weighed after leaving the weaning sheds. The specificity of a realtime PCR amplifying the infB gene was evaluated with 68 H. parasuis isolates and 36 strains of closely related species. 239 samples of DNA from tissues and fluids of 16 experimentally challenged animals were also tested with the realtime PCR, and the results compared with culture and a conventional PCR. The farm experiments showed that none of the controlled challenge pigs showed any signs of illness due to Glasser's disease, although the treatment groups required more antibiotics than the controls. A total of 556 H. parasuis isolates were genotyped, while 150 isolates were serotyped. H. parasuis was detected on 19 of 20 farms, including 2 farms with an extensive history of freedom from Glasser's disease. Isolates belonging to serovars regarded as potentially pathogenic were obtained from healthy pigs at weaning on 8 of the 10 farms with a history of Glasser's disease outbreaks. Sampling 213 sick pigs yielded 115 isolates, 99 of which belonged to serovars that were either potentially pathogenic or of unknown pathogenicity. Only 16 isolates from these sick pigs were of a serovar known to be non-pathogenic. Healthy pigs also had H. parasuis, even on farms free of Glasser's disease. The realtime PCR gave positive results for all 68 H. parasuis isolates and negative results for all 36 non-target bacteria. When used on the clinical material from experimental infections, the realtime PCR produced significantly more positive results than the conventional PCR (165 compared to 86).

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

Interactions between rotation breaks, tillage and N management on sugarcane grown at Bundaberg and Ingham.

The impact of cropping histories (sugarcane, maize and soybean), tillage practices (conventional tillage and direct drill) and fertiliser N in the plant and 1st ratoon (1R) crops of sugarcane were examined in field trials at Bundaberg and Ingham. Average yields at Ingham (Q200) and Bundaberg (Q151) were quite similar in both the plant crop (83 t/ha and 80 t/ha, respectively) and the 1R (89 t/ha v 94 t/ha, respectively), with only minor treatment effects on CCS at each site. Cane yield responses to tillage, break history and N fertiliser varied significantly between sites. There was a 27% yield increase in the plant crop from the soybean fallow at Ingham, with soybeans producing a yield advantage over continuous cane, but there were no clear break effects at Bundaberg - possibly due to a complex of pathogenic nematodes that responded differently to soybeans and maize breaks. There was no carryover benefit of the soybean break into the 1R crop at Ingham, while at Bundaberg the maize break produced a 15% yield advantage over soybeans and continuous cane. The Ingham site recorded positive responses to N fertiliser addition in both the plant (20% yield increase) and 1R (34% yield increase) crops, but there was negligible carryover benefit from plant crop N in the 1R crop, or of a reduced N response after a soybean rotation. By contrast, the Bundaberg site showed no N response in any history in the plant crop, and only a small (5%) yield increase with N applied in the 1R crop. There was again no evidence of a reduced N response in the 1R crop after a soybean fallow. There were no significant effects of tillage on cane yields at either site, although there were some minor interactions between tillage, breaks and N management in the 1R crop at both sites. Crop N contents at Bundaberg were more than 3 times those recorded at Ingham in both the plant and 1R crops, with N concentrations in millable stalk at Ingham suggesting N deficiencies in all treatments. There was negligible additional N recovered in crop biomass from N fertiliser application or soybean residues at the Ingham site. There was additional N recovered in crop biomass in response to N fertiliser and soybean breaks at Bundaberg, but effects were small and fertiliser use efficiencies poor. Loss pathways could not be quantified, but denitrification or losses in runoff were the likely causes at Ingham while leaching predominated at Bundaberg. Results highlight the complexity involved in developing sustainable farming systems for contrasting soil types and climatic conditions. A better understanding of key sugarcane pathogens and their host range, as well as improved capacity to predict in-crop N mineralisation, will be key factors in future improvements to sugarcane farming systems.