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.

Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity

Fusarium wilt of banana is a potentially devastating disease throughout the world. Options for control of the causal organism, Fusarium oxysporum f.sp. cubense (Foc) are limited. Suppressive soil sites have previously been identified where, despite the presence of Foc, Fusarium wilt does not develop. In order to understand some aspects of this disease suppression, endophytic Fusarium oxysporum isolates were obtained from banana roots. These isolates were genetically characterized and compared with an isolate of Fusarium oxysporum previously identified as being capable of suppressing Fusarium wilt of banana in glasshouse trials. Three additional isolates were selected for glasshouse trials to assess suppression of Fusarium wilt in two different cultivars of banana, Cavendish and Lady Finger. One isolate (BRIP 29089) was identified as a potential biocontrol organism, reducing the disease severity of Fusarium wilt in Lady Finger and Cavendish cultivars. Interestingly, one isolate (BRIP 45952) increased Fusarium wilt disease severity on Cavendish. The implications of an isolate of Fusarium oxysporum, non-pathogenic on banana, increasing disease severity and the potential role of non-pathogenic isolates of Fusarium oxysporum in disease complexes are discussed.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Multidrug-resistant extraintestinal pathogenic Escherichia coli of sequence type ST131 in animals and foods.

Multidrug-resistant Escherichia colt sequence type 131 (51131) has recently emerged as a globally distributed cause of extraintestinal infections in humans. Diverse factors have been investigated as explanations for ST131's rapid and successful dissemination, including transmission through animal contact and consumption of food, as suggested by the detection of ST131 in a number of nonhuman species. For example, ST131 has recently been identified as a cause of clinical infection in companion animals and poultry, and both host groups have been confirmed as faecal carriers of ST131. Moreover, a high degree of similarity has been shown among certain ST131 isolates from humans, companion animals, and poultry based on resistance characteristics and genomic background and human and companion animal ST131 isolates tend to exhibit similar virulence genotypes. However, most ST131 isolates from poultry appear to possess specific virulence genes that are typically absent from human and companion animal isolates, including genes associated with avian pathogenic E. coli. Since the number of reported animal and food-associated ST131 isolates is quite small, the role of nonhuman host species in the emergence, dissemination, and transmission of ST131 to humans remains unclear. Nevertheless, given the profound public health importance of the emergent ST131 clonal group, even the limited available evidence indicates a pressing need for further careful study of this significant question.

Stem cankers on sunflower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe (Phomopsis) species.

The identification of Diaporthe (anamorph Phomopsis) species associated with stem canker of sunflower (Helianthus annuus) in Australia was studied using morphology, DNA sequence analysis and pathology. Phylogenetic analysis revealed three clades that did not correspond with known taxa, and these are believed to represent novel species. Diaporthe gulyae sp. nov. is described for isolates that caused a severe stem canker, specifically pale brown to dark brown, irregularly shaped lesions centred at the stem nodes with pith deterioration and mid-stem lodging. This pathogenicity of D. gulyae was confirmed by satisfying Koch's Postulates. These symptoms are almost identical to those of sunflower stem canker caused by D. helianthi that can cause yield reductions of up to 40% in Europe and the USA, although it has not been found in Australia. We show that there has been broad misapplication of the name D. helianthi to many isolates of Diaporthe ( Phomopsis) found causing, or associated with, stem cankers on sunflower. In GenBank, a number of isolates had been identified as D. helianthi, which were accommodated in several clades by molecular phylogenetic analysis. Two less damaging species, D. kochmanii sp. nov. and D. kongii sp. nov., are also described from cankers on sunflower in Australia.

A checklist of plant pathogenic and other microfungi in the rainforests of the wet tropics of northern Queensland

Microfungi that cause disease or are associated with diseased plants in the wet tropics of northern Queensland are listed. A total of 206 host-pathogen combinations on 148 host species has been compiled from the results of plant disease surveys in the Wet Tropics World Heritage Area in 1992 and 1993, from herbarium records and from previously published host-pathogen combinations.

Pathogenic variation of Alternaria species associated with leaf blotch and fruit spot of apple in Australia

Four Alternaria species groups (A. longipes, A. arborescens, A. alternata/A. tenuissima and A. tenuissima/A. mali) are associated with leaf blotch and fruit spot of apple in Australia. There is no information on the variability of pathogenicity among the species and isolates within each species causing leaf blotch or fruit spot. We used a detached leaf assay and an in planta fruit inoculation assay to determine the pathogenicity and virulence of the four Alternaria species. Our results showed that isolates within the same species were not specific to either leaf or fruit tissue and showed great variability in pathogenicity and virulence, indicating cross-pathogenicity, which may be isolate dependent rather than species dependent. Generally, virulence of A. tenuissima and A. alternata isolates on leaf and fruit was higher than other species. Isolates of all species groups were pathogenic on leaves of different cultivars, but pathogenicity on fruit of different cultivars varied among isolates and species. Implications of our findings on prevalence of the diseases in different apple-producing regions in Australia and the development of targeted disease management of the diseases are discussed

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre

To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. Fluoroquinolone-resistant faecal E. coli isolates (n232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

Polyphasic characterization of four new plant pathogenic Phyllosticta species from China, Japan, and the United States

The black rot disease of Vitis species and other host genera of Vitacease is caused by Phyllosticta ampelicida and allied taxa which is considered to be a species complex. In this paper, we introduce four new species of Phyllosticta, including two from the P. ampelicida complex, based on a polyphasic characterization including disease symptoms and host association, morphology, and molecular phylogeny. The phylogenetic analysis was conducted based on the ribosomal internal transcribed spacer (ITS) region and a combined multi-locus alignment of the ITS, actin (ACT), partial translation elongation factor 1-alpha (TEF-1), and glyceraldehydes 3-phosphate dehydrogenase (GPDH) gene regions. Our study confirms the phylogenetic distinctions of the four new species, as well as their phenotypic differences with known species in the genus.

Coliforms in processed mango: Significance and control

The aims of this investigation were to enumerate coliforms in fresh mangoes, puree, cheeks, and cheeks-in-puree in order to determine the source of these organisms in the processed products, to determine methods for their control, and to identify coliforms isolated from cheeks-in-puree to determine whether they have any public health significance. Product from four processors was tested on two occasions. The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 × 103 and 5.4 × 104 cfu/g. Pasteurisation reduced the coliform count of raw puree to < 5 cfu/g. Forty-seven percent of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as Klebsiella pneumoniae using the ATB 32E Identification System. Klebsiella strains were tested for growth at 10 °C, faecal coliform response, and fermentation of -melizitose, to differentiate the three phenotypically similar strains, K. pneumoniae, K. terrigena and K planticola. Results indicated that 41% of K. pneumoniae isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern for these tests and were designated ‘psychrotrophic’ K. pneumoniae. Klebsiella pneumoniae counts of between 2.1 × 103 and 4.9 × 104 cfu/g were predicted to occur in the retail packs of mango cheeks-in-puree produced by the processors who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurisation of puree, should be taken in order to inactivate it

Induced Resistance: Potential for Control of Postharvest Diseases of Horticultural Crops

Fortunately, plants have developed highly effective mechanisms with which to defend themselves when attacked by potentially disease-causing microorganisms. If not, then they would succumb to the many pathogenic fungi, bacteria, viruses, nematodes and insect pests, and disease would prevail. These natural defence systems of plants can be deliberately activated to provide some protection against the major pathogens responsible for causing severe yield losses in agricultural and horticultural crops. This is the basis of what is known as ‘induced’ or ‘acquired’ disease resistance in plants. Although the phenomenon of induced resistance has been known amongst plant pathologists for over 100 years, its inclusion into pest and disease management programmes has been a relatively recent development, ie. within the last 5 years. This review will discuss very briefly some of the characteristics of the induced resistance phenomenon, outline some of the advantages and limitations to its implementation and provide some examples within a postharvest pathology context. Finally some approaches being investigated by the fruit pathology team at DPI Indooroopilly and collaborators will be outlined.

Presence of Nicotinamide Adenine Dinucleotide–Independent Haemophilus paragallinarum in Mexico

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent—growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3 wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NADindependent H. paragallinarum of serovar B has been reported. Abbreviations: NAD ¼ nicotinamide adenine dinucleotide; NAM ¼ nicotinamide; PCR ¼ polymerase chain reaction; TM ¼ complete growth medium without chicken serum or nicotinamide adenine dinucleotide; TM/SN ¼ complete growth medium that contains both chicken serum and nicotinamide adenine dinucleotide