The development of a rapid and reliable method (SAPSWash): For the extraction and recovery of spermatozoa from superabsorbent polymer containing products.

This work presents a rapid and reliable method to recover spermatozoa from Super Absorbent Polymers (SAPs) commonly found in sanitary protection products such as nappies and sanitary towels. The use of salt solutions was investigated and a protocol was developed using a calcium chloride (CaCl2) solution to release semen deposited onto a selected SAP containing product. The method was tested on ultra-sanitary towel samples treated with a known amount of semen. A range of treatments were examined; some samples were prepared and immediately frozen for storage and others were allowed to air dry overnight to replicate the condition of similar items recovered for examination in sexual offence cases. The method allowed the collection of low yields of spermatozoa, but these were still sufficient for microscopic identification of intact heads and to obtain ESI17 DNA profiles from all the samples. This report presents the method, the results obtained and discusses prospective adaptations to the method for validation to implement the method into forensic casework.

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori.

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

Microbial anaerobic demethylation and dechlorination of chlorinated hydroquinone metabolites synthesized by basidiomycete fungi.

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.

Detection and phylogenetic analysis of novel crenarchaeote and euryarchaeote 16S ribosomal RNA gene sequences from a Great Barrier Reef sponge.

The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges.

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl.

Anaerobic bacteria reductively dechlorinate polychlorinated biphenyls (PCBs) in aquatic sediments, but these microorganisms remain uncultured and, until now, unidentified. Through denaturing gradient gel electrophoresis (DGGE) of 16S rDNA from a highly enriched ortho-PCB dechlorinating culture, the growth of a single microorganism was shown to be dependent upon the presence and dechlorination of 2,3,5,6-tetrachlorobiphenyl. This is the first identification of a microorganism that catalyses the reductive dechlorination of a PCB. The organism, bacterium o-17, has high sequence similarity with the green non-sulphur bacteria and with a group that includes Dehalococcoides ethenogenes. Bacterium o-17 required acetate for dechlorination and growth. H2:CO2 (80:20 at 101 kPa) did not support dechlorination or growth of the dechlorinator. Archaeal 16S rDNA was not detected in actively dechlorinating bromoethanesulphonate-treated non-methanogenic cultures, which indicated that methanogenic Archaea were not required for dechlorination. The consistent association with dechlorinating activity combined with high similarity to other known dechlorinating microorganisms indicates that bacterium o-17 catalyses the reductive ortho-dechlorination of 2,3,5,6-tetrachlorobiphenyl.

Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques.

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).

Antibody response against Pseudomonas aeruginosa membrane proteins in experimentally infected sheep.

Shedded sheep inoculated epicutaneously with P. aeruginosa and then wetted experimentally by a sprinkler system, rapidly develop a green bacterial stain. This was associated with an outpouring of serious exudates onto the skin surface in the fleecerot lesion site. Histopathological analysis of dermatitic lesions revealed an infiltration of polymorphonuclear leucocytes into the dermis and the formation of a mosaic of microabscesses beneath the sloughed sheets of cornified epithelium. P. aeruginosa if present, was always localized as aggregates at the leading front of the seropurulent exudate and was never observed to invade the dermis. Animals that had been inoculated with P. aeruginosa but kept dry, showed no signs of dermatitis or serological reactivity against the inoculated bacterium. In contrast, sheep that had been inoculated and wetted, reacted serologically against P. aeruginosa whole cells in an enzyme-linked immunosorbent assay (ELISA). Eleven of 18 sheep were considered to be high-antibody responders and registered an ELISA ratio > 2.5 at one or more time points over the duration of the experiment (14 weeks). Analysis of ELISA reactivity of fleecerot sheep against fractionated cell envelope proteins of P. aeruginosa showed a preferential antibody response to outer (OMP) rather than inner (IMP) membrane proteins. Immunoblots revealed strong antibody activity against 2 major OMPs-Opr F and Opr H with apparent molecular masses of 39 and 21 kDa respectively. OMPs prepared from sarkosyl-resistant outer membrane vesicles were electrophoretically identical to OMPs prepared by a more rapid and efficient organic phase partitioning procedure (Chin and Dai, 1990). Although two other OMPs-Opr E (44 kDa) and Opr G (25 kDa) were seen in Coomassie blue-stained SDS-PAGE gels of P. aeruginosa OMPs, they were not reactive with sera from fleecerot affected sheep. It is likely that sheep with high levels of circulating serum antibody against major outer membrane proteins of P. aeruginosa may, in the event of a fleecerot episode, exude such antibodies onto the skin surface. This could provide a strategy for the control of ovine fleecerot by vaccination if highly conserved outer membrane proteins of P. aeruginosa were found to be protective.

Relationship between the immune response of sheep and the population dynamics of bacteria isolated from fleecerot lesions.

In sheep wetted by rain, proliferation of bacteria in the skin-fleece microenvironment invariably discolours the fleece and causes a dermatitic condition known as fleecerot. The changes in population dynamics of fleece bacteria were analysed by carrying out skin washings at randomly selected sites on the back of sheep before, and at 48 h and 96 h after exposure to rain. Gram-positive rods belonging to Bacillus species (10(2)-10(4) cfu/cm2) predominated in dry fleece. Gram-positive cocci (e.g. Micrococcus and Staphylococcus species) as well as Gram-negative rods (pseudomonads) were also present but in lower abundance (less than 10(2) cfu/cm2). Fleece bacterial populations generally increased in numbers during the first 24-48 h of wetting. By 96 h however, skin washings showed a preponderance of Pseudomonas aeruginosa (10(4)-10(6) cfu/cm2) and to a lesser extent, pigmented Micrococcus species. Growth of fleece bacteria was associated with a characteristic green or yellow/orange staining of fleece. Fewer species of bacteria were isolated from sheep showing green staining while those animals with yellow/orange discolourations appeared to have a more mixed microflora composition. The predominance of P. aeruginosa in the wet fleece of sheep displaying either green or yellow/orange bacterial stain, was accompanied by a significant serological response against this species. Since skin bacteria have never been observed to penetrate cutaneously in skin sections biopsied from fleecerot sites, it must be concluded that the sheep skin is sensitized by continuous exposure to antigens that are associated with or released by P. aeruginosa.

Comparative effectiveness of avermectins and deltamethrin in suppressing oviposition in Lucilia cuprina (Diptera: Calliphoridae).

The activities of three avermectins and deltamethrin as oviposition suppressants were investigated with a laboratory bioassay in which gravid females of the blowfly Lucilia cuprina (Wiedemann) were exposed to treated oviposition targets. An easily comparable index of suppression, the oviposition suppression concentration (OSC), was defined. All four compounds were effective oviposition suppressants. The three avermectins had similar OSC50 values (approximately 13 ppm). Deltamethrin, with an OSC50 of 0.4 ppm, was the most potent suppressant. The avermectins all produced significant mortality in adults with suppressed oviposition, while deltamethrin did not cause an increase in deaths at concentrations giving up to 100% suppression of oviposition. The toxicities of all four compounds to adult females were similar when assessed by topical application.

Dermal and serological response against Pseudomonas aeruginosa in sheep bred for resistance and susceptibility to fleece-rot.

Genetically select lines of Merino sheep have been bred at Trangie (NSW Agriculture and Fisheries) for resistance (R) or susceptibility (S) to fleece-rot and flystrike. It is believed that fleece characters are primarily responsible for the R or S phenotype. When transferred to the wetter coastal environment of Sydney, R and S sheep with no more than 6 weeks wool cover, continued to show significant differences in the incidence and severity of fleece-rot dermatitis. To test the hypothesis that these sheep might also exhibit differences in their local skin reactions and immune responsiveness, 3 intradermal injections of killed Pseudomonas aeruginosa were administered at monthly intervals. After primary intradermal challenge, R sheep had a higher incidence of skin induration and a stronger inflammatory response (increased induration diameter) than S sheep. Compared to S sheep, R sheep also developed higher levels of circulating antibodies against whole cell antigen and both inner and outer membrane proteins of P. aeruginosa. These responses were maintained in R sheep with each consecutive challenge while S sheep showed a decline in their immune responsiveness. Differences in antibody response against outer membrane proteins were also detected when antigenically naive sheep from each genetic line were sensitised by epicutaneous challenge with P. aeruginosa under experimental wetting conditions. Intradermal challenge of these animals 6 months later with outer membrane proteins, revealed a late maximum (72 h) in the development of induration diameters for R sheep while S animals showed maximal induration diameters by 24 h. However, there was no significant difference in induration response between 24 h and 72 h within each group of sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological properties of phospholipase C purified from a fleecerot isolate of Pseudomonas aeruginosa.

A role for one of many exocellular enzymes produced by Pseudomonas aeruginosa--phospholipase C (PLC)--as a prime candidate virulence factor in fleecerot dermatitis has been examined. The addition of Tween 80 in tryptose minimal medium effectively perturbed the membrane system of a field isolate of P. aeruginosa, resulting in increased production and release of a periplasmic enzyme marker, alkaline phosphatase (AP), and also of PLC. PLC activity levels in the culture supernatant were 10- to 15-fold higher in the presence of Tween than in its absence. Apart from AP, the culture medium contained little or no detectable proteolytic enzyme activity, thereby facilitating the partial purification of a haemolytic form of PLC by anion-exchange chromatography. This enzyme, when injected intradermally into the skin of sheep, elicited histopathological lesions virtually identical to those seen in naturally occurring fleecerot. In addition, serum from each of eight sheep afflicted with fleecerot contained high levels of circulating anti-PLC antibody activity when assayed by ELISA. Since these antibodies did not affect the enzymic function of PLC, it is likely that they do not bind to, or are incapable of conformational modification of, the active site.

Experimental production of dermatitis in sheep with Pseudomonas aeruginosa.

The attachment, to sheep skin, for 4 days, of control wool pads saturated with sterile culture medium which contained a bacteriostat, induced only a mild dermatitis, whereas wool pads saturated with Pseudomonas aeruginosa culture induced a subacute dermatitis characterised by scaling, microabscess formation and seropurulent exudate. Changes similar to the latter were observed in skin affected by natural fleece-rot which developed spontaneously after 7 days of artificial wetting and in which P. aeruginosa was the predominant species of bacteria. An exacerbatory, if not causal, role for this organism is suggested in the development of the dermatitis associated with fleece-rot and in the exudation of seropurulent material, a step essential in the development of body strike.

The ovipositional response of the Australian sheep blowfly, Lucilia cuprina, to fleece-rot odours.

The ovipositional response of Lucilia cuprina flies to odours emanating from fleece-rot lesions of greasy wool in which Pseudomonas aeruginosa bacteria proliferated, was studied. Fractionation of the fleece-rot odours was carried out by bubbling the volatile components through hydrochloric acid and sodium hydroxide solutions to remove basic odours and acidic odours respectively. It was found that the acidic/neutral odours of fleece-rot wool, when perfused into wet, greasy wool stimulated L. cuprina to oviposit. On the other hand, the basic/neutral odours of fleece-rot wool were virtually unattractive to the gravid fly. Similarly, the acidic/neutral odours emanating from fleece-rot lesions of clean wool from which the non-fibre components, wax, suint and epithelial debris, had been removed by scouring, were found to be unattractive to the gravid fly in choice tests.

Observations on fibre diameter variation of sheep in relation to fleece-rot and body strike susceptibility.

A comparative study of various fleece properties known to influence fleece-rot susceptibility was made in a merino flock consisting of sheep which were found to be either resistant or susceptible to fleece-rot and body strike following heavy rains. The fleece properties measured were fibre diameter, fibre diameter, fibre diameter variation, wax content, suint content, wax to suint ratio, suint pH, insoluble nitrogen content, wool colour and wettability. Fibre diameter variation, due mainly to the presence of coarse, secondary fibres in the staple, was the only fleece property which differed significantly (p less than 0.001) between resistant and susceptible animals. The coefficient of variation of fibre diameter was lowered from a mean value of 22.7 +/- 0.3% in susceptible sheep to 20.0 +/- 0.3% in resistant sheep. A causal relationship between high fibre diameter variation and fleece-rot susceptibility is suggested. Sheep with irregular fibre size may retain free moisture in the fleece for longer, and thereby become more susceptible to fleece-rot than sheep with uniform fibre diameter, other predisposing factors being equal.

The significance of certain skin characters of sheep in resistance and susceptibility to fleece-rot and body strike.

Micro-anatomical differences in skin structure associated with resistance and susceptibility of sheep to fleece-rot and body strike were identified, and found to be of similar magnitude in 2 genetically divergent flocks of medium-woolled Merino ewes. Susceptible sheep were characterised by smaller follicle groups, resulting in higher densities of follicle populations with greater concentrations of the primary follicles (and sudoriferous glands) than in the resistant sheep. From these smaller follicle groups of susceptible sheep, thicker wool fibres grew than were found in the resistant sheep. The significance of these findings is discussed in relation to the pathogenesis of fleece-rot and body strike of sheep, and the value of the measured skin characters as a method for identifying resistant sheep.

The blowfly strike problem of sheep in New South Wales.

A field survey was undertaken between 1972-76 to reappraise the nature of the blowfly problem in New South Wales. For 2 years, 1972-1974, some 80,000 sheep were kept under observation and 12,481 strikes were reported, most due to Lucilia cuprina. Breech strike was still the basic problem but tail strike associated with scouring had become an important component where pastures have been improved. Breech strike was controlled at minimal cost by managerial practices such as docking tails the correct length--second joint-space palpable ventrally (midway down the vulval orifice in ewes) for radically mulesed lambs and the third joint-space (tip of vulva in ewes) for all other lambs-mulesing at lamb marking, mid-season crutching, determining the cause of scouring and applying the appropriate preventative or remedial measures. Thus the use of insecticides could be reserved for the control of body strike in young sheep in the odd wet years and poll strike in horned rams. Major outbreaks of body strike occurred in 1973/74. Body strike worried graziers most because of its unpredictability, sudden onset and scale. and only failing insecticides were available for control.