Evaluation of the Effective Inactivation of Enteric Bacteria and Viruses From Swine Effluent and Sludge at Tropical Temperatures

The safe recycling of organic wastes such as the nutrients obtained from agriculture activities is a relevant aspect of the “One Health” strategy, a worldwide initiative including all aspects of health care for humans and animals, as well as aspects related to the conservation and development of the environment. It represents an important opportunity to mitigate the potential impact of microbial pathogens likely present in nutrient natural sources, as digestate, when applied to land as fertilizers. The objectives of the current study were to determine the inactivation of enteric microorganisms in swine digestate (effluent and sludge) during storage in anaerobic conditions and at different ambient temperatures of tropical zones (16, 22, and 37 °C). Human adenovirus 2 (HAdV-2), bacteriophage PhiX-174, and Salmonella enterica serovar typhimurium (S. typhimurium) were used as enteric microbial models to determine the minimum storage time required to reduce at least 3log of enteric microorganisms load in both matrices at the mentioned temperatures. The minimum storage time required for S. typhimurium was shorter than those observed for viruses at 37 °C, while reduction of at least 3log for S. typhimurium was observed after just 1 day; 20 and 90 days were needed for similar reductions for HAdV-2 and PhiX-174, respectively. Similar results were also observed at lower temperatures: 30 and 45 days were needed for S. typhimurium at 22 and 16 °C, respectively, but 30 and 90 days were needed at 22 °C for HAdV-2 PhiX-174, respectively, or 90 and 120 days at 16 °C for HAdV-2 PhiX-174, respectively. This is a pioneering study on the evaluation of inactivation of enteric viruses in swine digestate in usual tropical ambient temperatures; it demonstrates that the time for a considerable inactivation is longer than that observed for a enteric microorganisms (S. typhimurium), and shows that the storage of swine digestate at the referred temperatures can be used as a low-cost post-treatment to inactivate enteric microorganisms, allowing a further use as safer fertilizer.     

Real‐time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence

Fire blight is the most devastating bacterial disease of rosaceous plants. Forecasting fire blight infections is important to allow for countermeasures that reduce economic damage in pome fruit production. Current computerized forecasting models are solely based on physical factors such as temperature and moisture, but not on the actual presence of the pathogen Erwinia amylovora. Although the inoculum concentration is considered to be crucial for infection and disease outbreak, most current approaches used for identification of fire blight inoculum including morphological, biochemical, serological, and DNA‐based methods are nonquantitative. Based on a real‐time PCR approach previously published, an improved protocol to be used directly on whole bacteria in the field is described. The method allows for early detection and quantification of the pathogen prior to the occurrence of first symptoms. There is a clear correlation between bacterial abundance and subsequent disease development. However, in some cases, no disease symptoms could be observed despite a pathogen load of up to 3·4 × 10⁶ cells per blossom. Integration of the amount of pathogen detected into refined prediction algorithms may allow for the improvement of applied forecasting models, finally permitting a better abatement of fire blight.     

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Antimicrobial resistance of Staphylococcus aureus and coagulase negative staphylococci isolated from mastitis milk samples from sheep and goats

Staphylococcus aureus and coagulase negative staphylococci (CNS) were isolated from ovine and caprine mastitis milk samples originating from more than 40 Swiss farms. CNS dominated as causal microorganisms of mastitis in small ruminants. By restriction fragment length polymorphism (RFLP) analysis of the groEL gene and sequencing of 16S rDNA, various CNS species were identified, albeit certain of them predominated. For susceptibility testing of mastitis pathogens to selected antibiotics, minimal inhibitory concentrations were determined. Of the 67 S. aureus and 208 CNS strains, 31.3 % and 8.2 % were resistant to penicillin, 29.9 % and 1.0 % to ampicillin, 1.5 % and 10.6 % to erythromycin, and 3.0 % and 7.7 % to tetracycline, respectively. Moreover, 10 CNS strains (4.8 %) were resistant to oxacillin and one CNS strain to sulfamethoxazole/trimethoprim. The results obtained describe for the first time the resistance situation of mastitis pathogens from sheep and goats in Switzerland. However, accompanying and preventing measures are also of importance in mastitis control of small ruminants.     

Brucellosis associated with stillbirth in a bottlenose dolphin in Australia

A captive adult female bottlenose dolphin presented with stillbirth. The placenta appeared oedematous. No other gross lesions were evident in the placenta or the stillborn calf. Histopathology revealed mild multifocal placentitis and foetal encephalitis. Brucella sp. was isolated from lung, liver, spleen and kidney. Sequence and phylogenetic analysis demonstrated this organism to be most similar to Brucella ceti sequence type (ST) 27. Brucella sp. DNA was detected in formalin-fixed paraffin-embedded placenta and brain by real-time PCR using primers targeting the IS711 gene. Immunohistochemical staining revealed Brucella sp. antigen in placental inflammation. This is the first report of isolation of Brucella sp. from a marine mammal in the Southern Hemisphere and the first report of marine Brucella-associated disease in Australia.     

Polymers and polymer building blocks from meadowfoam oil

Meadowfoam oil methyl ester was metathetically cleaved with ethylene catalyzed by Re₂O₇–B₂O₃/Al₂O₃–SiO₂ + SnBu₄ or bis(tricyclohexyl phosphine) benzylidene ruthenium dichloride yielding -olefins, ,ω-dienes and 5-hexenoic acid methyl ester as the main product. The corresponding ω-epoxy fatty acid ester was achieved by chemoenzymatic epoxidation with H₂O₂ in the presents of Novozym^® 435. Glutaric acid mono methyl ester was synthesized by ruthenium-catalyzed oxidative cleavage with peracetic acid. Hydrocarboxylation with carbon monoxide/methanol yielded pimelic acid dimethyl ester. Metathetical dimerization of 5-hexenoic acid methyl ester yielded 5-decen-dioic acid dimethyl ester, which was subsequently polycondensed with ethylene glycole and 1,4-butanediol to high molecular weight polyesters. The ω-unsaturated ester was directly copolymerized with ethylene by an insertion-type palladium-catalyzed reaction to a high molecular weight functional polyolefin. The 5-epoxy hexanoic acid ester was polymerized to a comb like polyether.     

Genetic approach for the fast discovery of phenazine producing bacteria

A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures.     

Equine neonatal septicaemia: 24 cases

Equine neonatal septicaemia was confirmed in 24 foals hospitalised at the Rural Veterinary Centre between 1989 and 1992 with suspected septicaemia. Septicaemia was confirmed by culture of bacteria from blood of live foals and tissues obtained at necropsy of foals that died or were euthanased. Pathogenic bacteria isolated were predominantly Enterobacteriaceae (including Escherichia coli and Salmonella serovars) and Actinobacillus equuli. Clinical manifestations of septicaemia included signs of depression, dehydration, abnormalities in body temperature and manifestations of localised infection including diarrhoea, pneumonia, and septic arthritis. Most common haemato-logical abnormalities were neutropenia and increase of circulating band neutrophils. Survival rate of foals with confirmed septicaemia was 70.8%. Survival was found to be less likely in the presence of pneumonia, severe signs of depression, marked haematological changes or septic arthritis at the time of admission. Seven foals were confirmed to have septic arthritis without concurrent septicaemia. Of these, 4 had multiple joint involvement. Bacteria isolated from infected joints were predominantly Salmonella serovars. Four foals with septic arthritis failed to survive, due to multiple joint infection, which was unresponsive to treatment. The clinical and haematological abnormalities present in foals with confirmed septicaemia and septic arthritis were consistent with those observed in other studies. The bacterial isolates from foals with confirmed septicaemia were similar to those isolated in other studies. In contrast, the bacteria isolated from foals with septic arthritis without concurrent septicaemia were different from other studies.