Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Density and carrying capacity in the forgotten tigerland: Tigers in the understudied Nepalese Churia

While there are numerous wildlife ecology studies in lowland areas of Nepal, there are no in‐depth studies of the hilly Churia habitat even though it comprises 7642 km² of potential wildlife habitat across the Terai Arc. We investigated tiger, leopard and prey densities across this understudied habitat. Our camera trapping survey covered 536 km² of Churia and surrounding areas within Chitwan National Park (CNP). We used 161 trapping locations and accumulated 2097 trap‐nights in a 60‐day survey period during the winter season of 2010–2011. In addition, we walked 136 km over 81 different line transects using distance sampling to estimate prey density. We photographed 31 individual tigers, 28 individual leopards and 25 other mammalian species. Spatial capture–recapture methods resulted in lower density estimates for tigers, ranging from 2.3 to 2.9 tigers per 100 km², than for leopards, which ranged from 3.3 to 5.1 leopards per 100 km². In addition, leopard densities were higher in the core of the Churia compared to surrounding areas. We estimated 62.7 prey animals per 100 km² with forest ungulate prey (sambar, chital, barking deer and wild pig), accounting for 47% of the total. Based on prey availability, Churia habitat within CNP could potentially support 5.86 tigers per 100 km² but our density estimates were lower, perhaps indicating that the tiger population is below carrying capacity. Our results demonstrate that Churia habitat should not be ignored in conservation initiatives, but rather management efforts should focus on reducing human disturbance to support higher predator numbers.     

Effectiveness of Natural Compounds on DNA Damage in <Emphasis Type="Italic">Coris julis</Emphasis> (Linneaus 1758) from a Polluted Marine Area

The evaluation of DNA damage in aquatic organisms represents one of the most widely used biomarkers in the assessment and monitoring of marine pollution. Our previous research highlighted the presence of DNA damage in hepatic nuclei and blood cells of Coris julis specimens collected from Augusta harbor (Syracuse, Italy), a site highly polluted. In this work, we investigated on the ability of different natural compounds with antioxidant and immunostimulating properties, such as resveratrol (50 μM, 100 μM), amygdalin (100 μM, 200 μM), and Urtica dioica roots extract (50 and/or 100 μg/ml), in reducing DNA damage of C. julis. Blood cells were analyzed by atypical cellular comet assay. The results confirmed that Augusta specimens are the most damaged and showed that resveratrol, followed by amygdalin and U. dioica roots extract, drastically reduced DNA damage. This finding evidences the effectiveness of three natural compounds for DNA protection suggesting the possible use of feed enriched with antioxidant compounds in aquaculture practices for organisms damaged by natural and anthropic insults.     

DIC and granulomatous vasculitis in a dog with disseminated histoplasmosis

Disseminated histoplasmosis and disseminated intravascular coagulopathy were diagnosed in a 7 mo old, female spayed mixed-breed dog. The dog improved transiently with supportive care, but deteriorated shortly after initiation of antifungal therapy. The dog was subsequently euthanized. At necropsy, marked granulomatous vasculitis was identified in all affected organs. The tunicae and laminae of the arteries and arterioles were obscured by epithelioid macrophages and multinucleated giant cells admixed with necrotic material. Intracytoplasmic yeast were present within some of these macrophages. To the authors' knowledge, this is the first reported case of granulomatous vasculitis associated with Histoplasma capsulatum in a dog.     

Status of Biological Control of Plant Diseases with Composts

? Composts have the potential to provide biological control of plant diseases. Foliar as well as root pathogens may be affected. Many factors control these effects. Heat exposure during composting kills or inactivates pathogens if the process is monitored properly. Unfortunately, most biocontrol agents also are killed by this heat treatment. Thus, biocontrol agents must recolonize composts after peak heating. The raw feedstock, the environment in which the compost is produced, as well as conditions during curing and utilization determine the potential for recolonization by this microflora and disease suppression. Controlled inoculation of compost with biocontrol agents has proved necessary to induce consistent levels of suppression on a commercial scale. The decomposition level (stability) of composts also affects suppressiveness. Immature composts serve as food for pathogens and increase disease even when biocontrol agents are present. On the other hand, excessively stabilized organic matter does not support the activity of biocontrol agents. Composts also may induce detrimental effects. For example, chemical factors may negate suppressiveness. Salinity and the concentration of nitrogen in composts are principal chemical factors to be considered.     

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.     

Ivermectin toxicosis in a dog

A 5-year-old male Doberman Pinscher was examined after ingesting an equine paste dewormer containing approximately 115 mg of ivermectin. Clinical signs consisted of profound hypothermia, mild dehydration, dilated unresponsive pupils, localized muscle group fasciculations around the face and hind limbs, and no response to any external stimuli. Twelve days after parenteral administration of isotonic fluids and IV administration of dexamethasone and dimethyl sulfoxide, the dog returned to a clinically normal neurologic state. Ivermectin toxicosis has been reported frequently in Collies; however, other breeds may have idiosyncratic reactions to low doses. Patients with severe toxicosis should eventually recover completely if given appropriate intensive care.     

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.