Implications and challenges of tuberculosis in wildlife ungulates in Portugal: a molecular epidemiology perspective

Mycobacterium bovis and, more rarely, Mycobacterium caprae, may cause zoonotic bovine tuberculosis (bTB) in an extensive range of animal species. In Portugal, during 2009, a remarkable raise of bTB incidence was registered in cattle along with an increase of new cases in wildlife. In this work, we reassess and update the molecular epidemiology of bTB in wild ungulates by including 83 new M. bovis and M. caprae isolates from wild boar and red deer obtained during 2008-2009. Spoligotyping identified 27 patterns in wild ungulates, including 11 patterns exclusive from deer and five from wild boar. The genetic relatedness of wildlife and livestock isolates is confirmed. However, the relative prevalence of the predominant genotypes is different between the two groups. Contrasting with the disease in livestock, which is widespread in the territory, the isolation of bTB in wildlife is, apparently, geographically localized and genotypic similarities of strains are observed at the Iberian level.     

Hydrogen peroxide,iodine solution and methylene solution highly enhance the hatching rate of freshwater ornamental fish species

The aim of this study was to evaluate the effect of hydrogen peroxide, iodine solution (PVP) and methylene blue on eggs disinfection of three ornamental fish species, Danio rerio, Pterophyllum scalare and Gymnocorymbus ternetzi. The main idea was to create conditions to enhance the hatching rates. Eggs of each species were exposed to different concentrations of hydrogen peroxide (5, 10, 15 and 25 mg/L), PVP (0.25, 0.5, 0.75 and 1 mg/L) and methylene blue (0.5 1, 2 and 3 mg/L). The optimal doses ranged between species and chemicals: for G. ternetzi, the concentrations that high enhanced the hatching rate were 1 mg/L for the PVP treatment, 25 mg/L for the hydrogen peroxide treatment and 3 mg/L for methylene blue treatment; for P. scalare, the best results were achieved with 25 mg/L for hydrogen peroxide treatment and 3 mg/L for methylene blue treatment. By contrast, for all the different chemical did not increased the D. rerio hatching rate. Results showed that hydrogen peroxide and methylene blue are the most versatile, effective and safe to use in these species. On the other hand, PVP can be used but with many precautions due to very low safety margin. Results clearly show that the optimal concentration of chemicals for eggs disinfection is fish species dependent and it is completely wrong to extrapolate concentrations between different chemicals and fish species. Our study suggests that P. scalare can be used as a model in study of effectiveness of new chemicals with potential to disinfect water and increase hatching rates.     

Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis

The vectorial competence of the tick Rhipicephalus sanguineus is discussed in relation to the epidemiology of canine visceral leishmaniasis, taking into account its strict association with dogs and the low indices of natural infection presented by its known vector, the phlebotomine sand fly Lutzomyia longipalpis. In order to evaluate natural infection by Leishmania chagasi and the infectivity of these parasites in the tick, 39 specimens (6 females, 11 males and 22 nymphs) of R. sanguineus were removed from 21 dogs showing diverse symptoms of zoonotic visceral leishmaniasis (ZVL). Six ticks (15.4%) gave positive results for the genus Leishmania using the PCR technique. To determine the infectivity of the parasites, 36 hamsters were inoculated orally and peritoneally with macerates of ticks removed from nine dogs symptomatic for visceral leishmaniasis. After 6 months the hamsters were sacrificed and necropsied. Serum was removed for IFAT, as well as spleen and liver fragments to make imprint smears and for PCR. Eight (88.9%) of these dogs presented ticks that were infective for 14 hamsters (41.2%), 12 (85.7%) of them infected peritoneally and two (14.3%) orally. PCR revealed 27 smears (40.9%) to be positive, 20 (62.5%) of them infected peritoneally and seven (20.6%) orally. IFAT showed 14 positive animals (41.2%). Based on these findings, we suggest that the vectorial capacity of R. sanguineus for L. chagasi should be evaluated further, opening new perspectives in the epidemiology of ZVL.     

Control of postharvest diseases on citrus fruit by preharvest applications of biocontrol agent Pantoea agglomerans CPA-2: Part II. Effectiveness of different cell formulations

Infection of citrus fruit by postharvest pathogens often occurs in the field prior to harvest; therefore, it could be advantageous to apply biocontrol agents before harvest, which would reduce initial infection and then remain active and control pathogens in storage and under commercial conditions. The objective of the present work was to evaluate the effectiveness of different formulations of Pantoea agglomerans applied preharvest for controlling postharvest diseases on citrus. Results confirmed the protective effect of the additive Fungicover (FC) on populations of P. agglomerans exposed to non-conducive field conditions. In general, when osmotic-adapted and lyophilised P. agglomerans cells were used in bacterial treatments, these treatments showed greater survival rates than treatments with non-osmotic-adapted or fresh cells under field conditions. However, this superiority was only found when Fungicover was also added to suspensions of bacterial treatments. Therefore, bacterial treatments with Fungicover had population levels of P. agglomerans cells 1.2 and 2.8 log CFU cm⁻² higher than bacterial treatments without Fungicover during field experiments. These results allowed us to conclude that it is possible to improve environmental stress tolerance and ecological competence of P. agglomerans cells by integrating certain formulation strategies. Consequently, the improved formulation of P. agglomerans provided an effective control for orange fruit against natural postharvest pathogen infections and artificial infections of Penicillium digitatum with values of decay reduction higher than 50%. These latter results also demonstrated that it is possible to control postharvest pathogens using bacterial preharvest treatments.     

Metschnikowia andauensis as a new biocontrol agent of fruit postharvest diseases

Potential antagonists were isolated from the epiphytic flora associated with oranges and pome fruit. A total of 1465 microorganisms were tested in a preliminary screening against blue and green moulds on pome and citrus fruit, respectively. Among them, approximately 3% reduced incidence and severity by more than 50% and 4 microorganisms fulfilled the selection criteria of reduction in severity and incidence by 75%. The most effective was a yeast identified as Metschnikowia andauensis, strain NCYC 3728 (PBC-2), isolated from the surface of ‘Bravo de Esmolfe’ apple fruit cultivated in North Portugal. The biocontrol activity of M. andauensis PBC-2 was dependent on its applied concentration. At 5 × 10⁶ cfu/mL incidence (% of infected wounds) and severity (lesion diameter) were reduced by 62 and 70%, respectively and at 1 × 10⁷ cfu/mL, the greatest reduction was achieved, 90% of incidence and 95% of severity. The broad spectrum of action of M. andauensis PBC-2 was evaluated with effective control being achieved against Rhizopus stolonifer, Penicillium expansum and Botritys cinerea, on ‘Rocha’ pears and on different apple cultivars and against Penicillium digitatum and Penicillium italicum on mandarins and oranges. In semi-commercial trials in cold storage, the reduction of blue mould was 90%. Rapid colonization of fresh apple fruit wounds was observed during the first 24 h of cold storage, followed by a significant population increase during the first 15 days of storage and then the population remained stable until the end of storage.     

Ten-year regeneration responses to varying levels of overstory retention in two productive southern British Columbia ecosystems

We investigated survival and growth responses of planted and advance natural regeneration species of varying shade tolerance to partial retention harvesting in moist warm Interior Cedar-Hemlock (ICHmw2) and dry cool Montane Spruce (MSdk) ecosystems of southeastern British Columbia, Canada. Treatments included three levels of overstory basal area retention (none, light (∼25%), or heavy (∼50%)) installed by two harvest methods (handfelled or a pushover falling technique being tested for its ability to control the spread of root disease). After 10 years, growth of both planted and natural regeneration species of varying shade tolerance tended to increase with decreasing overstory retention and associated increases in light availability. In contrast, significant survival responses to retention level were lacking except in the case of shade-intolerant western larch. Harvest method had a variable effect on regeneration survival and growth. Where significant responses did occur, they were generally attributed to harvesting effects on the characteristics of planting microsites rather than root disease spread. Natural regeneration densities at the ICHmw2 site were high at all retention levels, whereas stocking was less consistent at the MSdk site. We concluded that moderately shade-tolerant to shade-tolerant interior spruce and western redcedar can, under conditions similar to those of our study sites, be successfully established under overstories of up to approximately 25 m²/ha basal area, but that growth performance is likely to be significantly lower than in clearcuts. Despite early survival issues, conclusions regarding Douglas-fir were similar. Poorer survival and vigour of shade-intolerant western larch suggested this species is not suitable for regeneration in partial retention systems where timber production is the primary objective. Where non-timber objectives predominate, survival and acceptable growth of even a small proportion of larch could add to the diversity of the regenerating stand.