Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.     

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.     

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.     

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Morphological and molecular analysis of Fusarium lateritium, the cause of gray necrosis of hazelnut fruit in Italy

Fusarium lateritium is a globally distributed plant pathogen. It was recently reported as the causal agent of nut gray necrosis (NGN) on hazelnut. Isolate characterization within F. lateritium was undertaken to investigate how morphological and molecular diversity was associated with host and geographic origin. Morphological studies combined with inter-simple-sequence repeat (ISSR) analysis, and phylogenetic analyses using translation elongation factor 1α (TEF-1α), β-tubulin genes, and nuclear ribosomal DNA internal transcribed spacer (ITS) sequences were conducted to resolve relationships among 32 F. lateritium isolates from NGN-affected hazelnut fruit, and 14 from other substrates or 8 from other hosts than hazelnut. Colonies of F. lateritium from hazelnut showed dark grayish-olive differing from the orange-yellow color of all other isolates from other hosts. Generally, isolates from NGN-affected fruit failed to produce sporodochia on carnation leaf agar. The influence of host and substrate on the genetic structure of F. lateritium was supported by ISSR and analyzed with principal coordinates analysis. A relationship between hazelnut and genetic variation was inferred. Phylogenetic analysis of ITS provided limited resolution while TEF-1α and β-tubulin analyses allowed a clear separation between the European and non-European F. lateritium isolates retrieved from GenBank, regardless of host. Though morphological traits of F. lateritium isolates from hazelnut were generally uniform in defining a typical morphogroup, they were not yet phylogenetically defined. In contrast, the typology related to slimy deep orange cultures, due to spore mass, grouped clearly separated from the other F. lateritium isolates and revealed a congruence between morphology and phylogeny.     

Effects of water deficit on the vegetative response,yield and oil quality of olive trees (Olea europaea L., cv Coratina) grown under intensive cultivation

An experiment was carried out in a young high-density olive grove (556 plants ha⁻¹—Olea europaea L., cv Coratina) located in Southern Italy to evaluate the effect of different soil water availability on the vegetative and productive performances of olive trees also looking into the quality of the resulting oils. Trials were carried out over a 3-year period on trees subjected to irrigation and grown under rainfed conditions. Vegetative tree response, as shoot elongation and trunk diameter, was evaluated. Yield per plant, fruit characteristics, oil quality indices (free fatty acid content, peroxide value, UV adsorption at 232 and 270 nm, total phenols, α-tocopherol content) were determined for both irrigated and non-irrigated treatments in the ‘on’ years 1997 and 1999 (6th and 8th year after planting, respectively).     

Canker and dieback on Platanus x acerifolia caused by Diaporthe scabra

Cankers and dieback on London plane caused by Diaporthe scabra have been observed in urban plantings in Catania, Italy. Symptoms were delayed spring flush, trunk cankers, small chlorotic leaves associated with dead terminal or lateral branches and an early defoliation. Isolations from infected wood on PDA led to the isolation of the anamorph Phomopsis scabra with α‐conidia production. The infected wood placed in a moist chamber developed pycnidia with α and β conidia and perithecia of the teleomorph D. scabra. Sequence analysis of the internal transcribed spacers ITS1 and ITS2, the ribosomal intragenic spacer beta‐tubulin 1 and beta‐tubulin 2 genes, congruently indicated that the D. scabra isolate is phylogenetically related to two strains of D. helianthi isolated in Italy from sunflower and to strains of D. eres. Inoculations on young plants of London plane growing in pots confirmed the pathogenicity of the isolated fungus. To our knowledge, this is the first report of the disease in Italy.