The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Growth requirements of some fungi causing maduromycosis

Three strains ofMadurella mycetomi, two ofM. grisea, and two ofRhinocladiella mansonii have been studied for ossible differences in growth requirements which might be used for distinguishing these species. Under the experimental conditions, an incubation temperature of 37C suitedM. mycetomi about as well as 30C.R. mansonii grew less well at 37C than at 30C, andM. grisea did not grow at the higher temperature. M. grisea andR. mansonii further differed fromM. mycetomi in that they required thiamine for growth. The pH tolerance of all the strains was very wide. Asparagine and potassium nitrate were readily utilized by all the strains, but ammonium salts were not. Urea was poorly used byM. mycetomi; the other species did not use it. A possible relationship ofM. grisea andR. mansonii is discusse

A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed-species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR-based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one-tube assay is simple, low-cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains.

Genetic diversity of subgroup 1 ilarviruses from eastern Australia

This is the first report of the genetic diversity within ilarvirus subgroup 1 from eastern Australia. It supports the separation of tobacco streak virus (TSV) strains from parthenium (Parthenium hysterophorus) and crownbeard (Verbescina encelioides) based on serology and host specificity. It has confirmed one previously described strain of TSV as a member of the species Strawberry necrotic shock virus and another as a new subgroup 1 ilarvirus, ageratum latent virus (AgLV), from Ageratum houstonianum. A multiplex RT-PCR showed that the genetically distinct strains of TSV and AgLV were commonly found in symptomless infections in virus-specific alternative weed hosts growing over a wide geographical range in eastern Australia. TSV has been one of the most damaging viruses in Australian oilseed and pulse crops in recent years, and this study has provided the taxonomic knowledge essential for the development of control programs for these viruses. © 2013 Springer-Verlag Wien.

Cotton bunchy top virus and other relatives

Cotton bunchy top virus (CBTV) and the related Cotton leafroll dwarf virus (CLRDV) have caused sporadic disease outbreaks in most cotton regions of the world. Until recently, little was known about the diversity of CBTV or its natural host range. Seven natural field hosts and one experimental host of CBTV have now been identified. These include cotton, Malva parviflora (Marshmallow weed), Abutilon theophrasti (Velvetleaf), Anoda cristata (Spurred anoda), Hibiscus sabdariffa (Rosella), Sida rhombifolia (Paddy’s lucerne), Chamaesyce hirta (Asthma plant) and Gossypium australe. These are currently the only eight known hosts of CBTV. However the virus may have a wider host range than originally thought and include further non-Malvaceae species like asthma plant (family Euphorbiaceae). There are two distinct strains of CBTV in Australia, -A and -B, which have been detected in cotton from numerous locations across almost all growing regions. From 105 samples of cotton that have been positive for CBTV, 6 were infections of strain A only, 60 were strain B only and 64 were a mixed infection of strains A and B. These results indicate the symptoms of cotton bunchy top disease are closely associated with the presence of strain CBTV-B. A diagnostic assay for Cotton leafroll dwarf virus (CLRDV - cotton blue disease) is being developed and applied successfully for the detection of CLRDV samples from Brazil and Thailand. This is the first confirmation of CLRDV from SE-Asia, which may pose an increased biosecurity threat to the Australian industry.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Variable-number tandem repeats genotyping used to aid and inform management strategies for a bovine Johne's disease incursion in tropical and subtropical Australia

The application of variable-number tandem repeats (VNTR) genotyping of Mycobacterium avium subsp. paratuberculosis isolates to assist in investigating incidents of bovine Johne’s disease in a low-prevalence region of Australia is described in the current study. Isolates from a response to detection of bovine Johne’s disease in Queensland were compared with strains from national and international sources. The tandem application of mycobacterial interspersed repetitive unit (MIRU) and multilocus short sequence repeats (MLSSR) genotyping identified 2 strains, 1 that infected cattle on multiple properties with trace-forward histories from a common infected property, and 1 genotypically different strain recovered from a single property. The former strain showed an identical genotype to an isolate from India. Neither strain showed a genotypic link to regions of Australia with a higher prevalence of the disease. Genotyping has indicated incursions from 2 independent sources. This intelligence has informed investigations into potential routes of entry and the soundness of ongoing control measures, and supported strategy and policy decisions regarding management of Mycobacterium avium subsp. paratuberculosis incursions for Queensland.

Do Phosphine Resistance Genes Influence Movement and Dispersal Under Starvation?

Phosphine resistance alleles might be expected to negatively affect energy demanding activities such as walking and flying, because of the inverse relationship between phosphine resistance and respiration. We used an activity monitoring system to quantify walking of Rhyzopertha dominica (F.) and a flight chamber to estimate their propensity for flight initiation. No significant difference in the duration of walking was observed between the strongly resistant, weakly resistant, and susceptible strains of R. dominica we tested, and females walked significantly more than males regardless of genotype. The walking activity monitor revealed no pattern of movement across the day and no particular time of peak activity despite reports of peak activity of R. dominica and Tribolium castaneum (Herbst) under field conditions during dawn and dusk. Flight initiation was significantly higher for all strains at 28 degrees C and 55% relative humidity than at 25, 30, 32, and 35 degrees C in the first 24 h of placing beetles in the flight chamber. Food deprivation and genotype had no significant effect on flight initiation. Our results suggest that known resistance alleles in R. dominica do not affect insect mobility and should therefore not inhibit the dispersal of resistant insects in the field.

Ecology, Behaviour and Control of Apis cerana with a Focus on Relevance to the Australian Incursion

Apis cerana Fabricius is endemic to most of Asia, where it has been used for honey production and pollination services for thousands of years. Since the 1980s, A. cerana has been introduced to areas outside its natural range (namely New Guinea, the Solomon Islands, and Australia), which sparked fears that it may become a pest species that could compete with, and negatively affect, native Australian fauna and flora, as well as commercially kept A. mellifera and commercial crops. This literature review is a response to these concerns and reviews what is known about the ecology and behaviour of A. cerana. Differences between temperate and tropical strains of A. cerana are reviewed, as are A. cerana pollination, competition between A. cerana and A. mellifera, and the impact and control strategies of introduced A. cerana, with a particular focus on gaps of current knowledge.

Gut-inhabiting yeasts from the gut of Australian passalid beetles

Passalidae (Polyphaga, Coleoptera) is a family of beetles with approximately 960 species that are distributed worldwide. Preliminary studies that characterized ascomycete and basidiomycete yeasts in the gut of these wood-eating beetles from the USA, Guatemala, and Thailand, demonstrated associations between certain yeast taxa and passalids. We extended the study to include yeasts and beetles from tropical forests near Cairns and Brisbane, Queensland, Australia. We isolated more than 1000 yeast strains from about 150 beetles belonging to 10 species. LSU and ITS rRNA markers were used to identify a subset of 250 yeast strains, which revealed that the gut of Australian passalids contained undescribed ascomycetes in the Debaryomyces, Dipodascus, Kazachstania, Ogataea, Scheffersomyces, Sugiyamaella, Spathaspora, Torulaspora, and Zygowilliopsis clades, as well as basidiomycetes in the genera Cryptococcus and Trichosporon. A close relative of Candida subhashii (Spathaspora clade) and the xylose-fermenting yeast Scheffersomyces stipitis were the most common species isolated in Queensland. These results agree with those of previous studies that showed a common association of xylose-fermenting yeasts in the gut of lignicolous insects. Species and higher taxa of yeasts, however, vary between Queensland passalids and those previously collected in distant regions.

Studies on an enzyme reacting with isoniazid fromMycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis H37Rv possesses an enzyme (referred to as ‘Y enzyme’) which catalyses in the presence of INH and NAD, the formation of a product, which turns yellow on acidification. The requirements for the reaction, such as enzyme concentration, INH concentration, etc., have been standardized. The substrate specificity of the enzyme with respect to INH and NAD has been determined. The reaction is specific for the INH-sensitive strain and is totally absent in INH-resistant strains. Furthermore, the ‘Y enzyme’ shows some characteristic features of a peroxidase in its requirement for oxygen and sensitivity to inhibition by various reagents. The requirements of this enzyme which is involved in the action of isoniazid inM. tuberculosis H37Rv is described for the first time.

Prevalence and potential fitness cost of weak phosphine resistance in Tribolium castaneum (Herbst) in eastern Australia

The prevalence of resistance to phosphine in the rust-red flour beetle, Tribolium castaneum, from eastern Australia was investigated, as well as the potential fitness cost of this type of resistance. Discriminating dose tests on 115 population samples collected from farms from 2006 to 2010 showed that populations containing insects with the weakly resistant phenotype are common in eastern Australia (65.2 of samples), although the frequency of resistant phenotypes within samples was typically low (median of 2.3). The population cage approach was used to investigate the possibility that carrying the alleles for weak resistance incurs a fitness cost. Hybridized populations were initiated using a resistant strain and either of two different susceptible strains. There was no evidence of a fitness cost based on the frequency of susceptible phenotypes in hybridized populations that were reared for seven generations without exposure to phosphine. This suggests that resistant alleles will tend to persist in field populations that have undergone selection even if selection pressure is removed. The prevalence of resistance is a warning that this species has been subject to considerable selection pressure and that effective resistance management practices are needed to address this problem. The resistance prevalence data also provide a basis against which to measure management success.

Evaluation and histological examination of a Campylobacter fetus subsp. venerealis small animal infection model

Bovine genital campylobacteriosis (BGC), caused by Campylobacter fetus subsp. venerealis, is associated with production losses in cattle worldwide. This study aimed to develop a reliable BGC guinea pig model to facilitate future studies of pathogenicity, abortion mechanisms and vaccine efficacy. Seven groups of five pregnant guinea pigs (1 control per group) were inoculated with one of three strains via intra-peritoneal (IP) or intra-vaginal routes. Samples were examined using culture, PCR and histology. Abortions ranged from 0 to 100 and re-isolation of causative bacteria from sampled sites varied with strain, dose of bacteria and time to abortion. Histology indicated metritis and placentitis, suggesting that the bacteria induce inflammation, placental detachment and subsequent abortion. Variation of virulence between strains was observed and determined by culture and abortion rates. IP administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising small animal model for the investigation of BGC abortion.

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.

Development of type I genetic markers from expressed sequence tags in highly polymorphic species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: - 1) priming site polymorphism in the template leads to inferior or erratic amplification; - 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions, and; - 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.