Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum)

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.     

Use of sentinel lambs for early monitoring of the South Powys Hydatidosis Control Scheme: prevalence of Echinococcus granulosus and some other helminths.

Sentinel lambs were used to identify young Echinococcus granulosus infections in sheep, to provide an early indication of the progress of the South Powys Hydatidosis Control Scheme. Four sentinel lambs were purchased on each of 60 farms, from inside and outside the control area; they were examined when approximately six, 10 and 15 months of age. Gross examination, thin slicing of organs and histological examination of the lesions in the viscera revealed no E granulosus hydatid cysts in lambs born within the control area, whereas 25 per cent of the 15-month-old lambs from outside the area harboured E granulosus cysts (less than 1 to 2 mm in diameter). Lambs from E granulosus infected farms had significantly higher anti-E granulosus ELISA antibody titres than lambs from uninfected farms. It was concluded that within one year of beginning to treat dogs with praziquantel every six weeks the transmission of E granulosus to sheep had ceased. In contrast, this treatment did not prevent infections with Taenia hydatigena or T ovis; an examination of the 240 lambs revealed T hydatigena in 33.3 per cent of them, Tovis in 4.2 per cent, Dictyocaulus filaria in 12.1 per cent and Meullerius capillaris in 49.2 per cent.     

Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.

Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.     

Microbial C and N dynamics during mesophilic and thermophilic incubations of ryegrass

Laboratory studies were conducted to determine C and N dynamics during the decomposition of ryegrass straw under mesophilic and thermophilic conditions. A K_C of 0.61 was developed for the chloroform-fumigation extraction method to estimate microbial biomass C. These estimates showed that the C and N requirement of the thermophilic biomass was approximately 50% of the mesophilic biomass. There was no relationship between chloroform-fumigation microbial biomass estimates and plating of microorganisms from straw on specific media. Mineralized C was measured as 185 and 210 g kg^-1 straw in the 25°C and 50°C treatments, respectively. The efficiency of microbial substrate use, on a total straw basis, was 34 and 28% in the 25°C and 50°C incubations, respectively. The level of soluble C declined more slowly than total C mineralization at both temperatures, indicating that a portion of the labile C was not readily biodegradable. The addition of N decreased the rate of C mineralization at both temperatures. The reduced N requirement of the thermophiles explains why rapid degradation of the high C:N residue occurred without additional N or the need for the addition of a low C:N ratio substrate. Additional inoculum did not affect the decomposition process. We conclude that the promotion of thermophilic biomass activities, through composting for example, may prove useful in upgrading agricultural wastes for introduction into sustainable cropping systems.     

Bat activity on riparian zones and upper slopes in Australian timber production forests and the effectiveness of riparian buffers

Unlogged buffers are used to ameliorate impacts to riparian areas in timber production forests. One function of these buffers is to protect the biodiversity of riparian areas. We measured bat activity in buffered streams with ultrasonic detectors across four different stream orders in logged, regrowth and mature forests (60 sites). Bat activity, foraging rates and species richness were similar in buffered streams surrounded by logged, regrowth and mature forests, suggesting that riparian areas effectively provide habitat for foraging and commuting bats in selectively logged forests. Vespadelus pumilus was the only species that responded to logging history, with decreased activity in mature forests. We found higher activity on larger rather than smaller order streams, a pattern also not affected by logging history. Bat activity along paired forest trail flyways on upper slopes (60 sites) was measured simultaneously with riparian flyway activity (for a total of 120 sites) to determine the importance of riparian areas relative to other available flyways. Activity was higher on upper slopes than on small streams, but similar to levels on larger streams. Total foraging activity was similar between riparian zones and upper slopes. Upper slopes contained higher species diversity, with Chalinolobus gouldii, Miniopterus schreibersii, Mormopterus norfolkensis, Scotorepens sp., Vespadelus pumilus and, to a lesser extent, Vespadelus darlingtoni detected more often than along streams. Other species (Rhinolophus megaphyllus, Nyctophilus spp. and Miniopterus australis) were not affected by topography. Estimates of total vegetation cover and, in particular, rainforest cover, were negatively associated with bat activity, highlighting the need for management of forest ‘clutter’ in regrowth forests for a suite of bat species. Streams and forest trails provide areas of lower clutter, which assist in maintaining high species diversity in regrowth forests. Our results support the use of riparian buffers, and point to the need for greater recognition of tracks on upper slopes as important habitat.     

The potential aboveground carbon storage of north American forests

To assess the possibility of using C offset as a method of sequestering CO₂ produced by the burning of fossil fuels, it is necessary to have accurate estimates of C reservoirs and fluxes. Recent studies have shown that estimates of C commonly used in the past are too large, and this may lead to confusion about the global C budget. Field data used in recent estimates of present C storage for the North American boreal and eastern deciduous forest biomes were reanalyzed to estimate their maximum potential C storage. The original data were collected using a stratified two-stage cluster survey sampling design. The reanalysis suggests that the boreal forest and eastern deciduous forest could sequester possibly as little as 13.4% (3.0 Pg) and 18.5% (1.5 Pg), respectively, more C than they presently store. These estimates represent the potential increase in C storage under present conditions, if the study areas were allowed to revert back into forests.     

Analysis of 2-methylisoborneol and geosmin in catfish by microwave distillation--solid-phase microextraction

The semivolatile cyclic alcohols 2-methylisoborneol (MIB) and geosmin (GSM) impart muddy or musty flavors to water and food products. A rapid quantitative analytical technique has been developed whereby microwave distillation is used to remove the volatile organic compounds from a lipophilic matrix into an aqueous matrix. Solid-phase microextraction (MD-SPME) is then used to extract and concentrate the analytes, which are then desorbed in the injection port of a gas chromatograph/mass spectrometer (GC/MS) for analysis. Limits of detection are 0.01 microg/kg and limits of quantification are 0.1 microg/kg. MD-SPME is comparable in precision, requires no solvents, and is faster than current methods of analysis. This methodology allows detection of MIB and GSM at concentrations below human sensory thresholds in fish tissue.