Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia.

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.     

Water column production and nutrient characteristics in mangrove creeks receiving shrimp farm effluent

Water column processes in undisturbed mangrove creeks and in creeks receiving effluent from shrimp farms in North Queensland, Australia, were studied. Small scale discharges into tidal creeks did not elevate dissolved nutrient concentrations compared with non‐impacted creeks, but did elevate concentrations of particulate nutrients, chlorophyll and suspended solids proximal to the site of the effluent discharge. Rates of primary and bacterial production downstream from the discharge exceeded rates in the shrimp ponds because of the synergistic effects of turbulent mixing and eutrophication. In the lower reaches of the mangrove creeks and immediately offshore, standing stocks of particulate material and rates of primary and bacterial production were within the range of values found in non‐discharge areas. Our results suggest that water column production by phytoplankton and bacteria strips nutrients from shrimp farm effluent and repackages them in more bioavailable forms.     

Rapid identification of virulence genes in enterotoxigenic Escherichia coli isolates associated with diarrhoea in Queensland piggeries

OBJECTIVE: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in southeast Queensland. DESIGN: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. PROCEDURE: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. RESULTS: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. CONCLUSION: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.     

Biofilm production by pathogens associated with canine otitis externa,and the antibiofilm activity of ionophores and antimicrobial adjuvants

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N‐acetylcysteine (NAC), Tris‐EDTA and disodium EDTA) were investigated spectrophotometrically (OD_570nm) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S. pseudintermedius clinical isolates were weak biofilm producers. 19/20 P. aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000–10,000 µg/ml and from 20,000–80,000 µg/ml against S. pseudintermedius and P. aeruginosa, respectively. Tris‐EDTA eradicated P. aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800 µg/ml. The MBEC was up to 16‐fold and eightfold higher than the MIC/MBC of NAC and Tris‐EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470 µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris‐EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm‐associated OE in dogs.     

Performance and environmental effects of forage production on sandy soils. II. Impact of defoliation system and nitrogen input on nitrate leaching losses

A field experiment was conducted over a 4‐year period to determine NO₃ leaching losses from grassland on a freely draining sandy soil. The experiment consisted of all combinations of five defoliation systems; cutting‐only (CO), rotational grazing (GO), mixed systems with one (MSI) or two silage cuts (MSII) plus subsequent rotational grazing, and simulated grazing (SG), four mineral nitrogen (N) application rates (0, 100, 200, and 300 kg N ha^?1 year^?1), and two slurry levels (0 and 20 m³ slurry ha^?1 year^?1). Due to the high N return by grazing animals, leaching losses in the rotational grazing systems generally were associated with NO₃‐N concentrations which exceeded the EU limit for drinking water. NO₃ leaching losses in a rotational grazing system could be reduced by lowering the N fertilizer intensity and the inclusion of one or two silage cuts in spring. However, even in the unfertilized mixed systems, N fixation by white clover exceeded the amounts of N removed via animal products, which resulted in NO₃‐N concentrations well above the EU limit for drinking water. In terms of leaching losses, the cutting‐only system was the most advantageous treatment. NO₃ leaching losses on grassland could be predicted by the amount of soil mineral N at the end of the growing season and by the N surplus calculated from N balances at the field scale. From the results obtained a revised nitrogen fertilization policy and a reduced grazing intensity by integrating silage cuts are suggested.     

Performance and environmental effects of forage production on sandy soils. III. Energy efficiency in forage production from grassland and maize for silage

Based on experimental data gathered in a research project on nitrogen fluxes in intensive dairy farming in Northern Germany, an analysis of fossil energy input and energy efficiency in forage production from permanent grassland and maize for silage was conducted. Field experiments comprised different defoliation systems and different rates of mineral N fertilizer and slurry application. Each change from grazing to cutting in grassland systems reduced the energy efficiency. Energy efficiency consistently decreased with increasing rates of mineral N application. In the production of maize for silage, maximum energy efficiency was obtained with an application of 50 kg N ha^?1 from slurry only. Net energy yields of maize for silage were much higher than that of grassland when compared at the same level of fossil energy and nitrogen fertilizer input. Considering both nitrate‐leaching losses and a necessary minimum quantity of grass herbage in a well‐balanced ration, it is suggested that a high proportion of maize for silage in combination with N‐unfertilized grass/clover swards used in a mixed cutting/grazing system represents a good trade‐off between the leaching of nitrates and energy efficiency.