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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. [Copyright &y& Elsevier]

Abstract: Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmania infantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis. [Copyright &y& Elsevier]

Stray cats in the city of Milan, Italy, were tested for Leishmania infantum and other selected infections. Twenty-seven cats (30.0%) were seroreactive by indirect fluorescent antibody test (IFAT), with an antibody titer of 1:40 for 16 (17.7%) cats and 1:80 (cut-off for feline L. infantum infection) for 11 (12.2%) cats. One blood (1.1%) and one popliteal lymph node (1.1%) sample tested positive by real-time polymerase chain reaction; no oculoconjunctival swabs tested positive. Feline immunodeficiency virus, feline leukemia virus, and feline coronavirus (FCoV) seroprevalence determined by enzyme-linked immunosorbent assay was 6.1, 6.1, and 39.0%, respectively. Toxoplasma gondii , Bartonella henselae , and Chlamydophila felis prevalence determined by IFAT was 29.3, 17.1, and 17.1%, respectively. The frequency of seroreactivity to L. infantum was significantly higher in FCoV-seropositive cats (OR = 4.4, P = 0.04). L. infantum -infected stray cats in Milan have a high seropositivity rate, comparable to that of cats in areas endemic for leishmaniosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Feline Leishmania strains are phenotypically similar to those from humans and dogs. • Feline, canine, and human strains have an identical susceptibility to drugs. • Parasites isolated from cats can infect feline, canine, and human macrophages. • Feline macrophages are permissive to strains isolated from dogs and humans. Zoonotic visceral leishmaniosis is a worldwide severe disease caused by Leishmania infantum , a protozoan that has phlebotomine sand flies as vectors and dogs as primary reservoir hosts. Over the last few decades, cats have been regarded as an indisputable piece within the ecological system in which L. infantum is maintained indefinitely. However, little is known about feline strains, including their phenotypic plasticity and infectivity. In this study, the phenotypic behaviour of seven L. infantum feline strains was compared to those of well-characterised counterparts isolated from two dogs and two humans in terms of growth profile, adaptive capacity under several stress conditions, susceptibility to antileishmanial drugs, and infectivity to host cells. Feline strains displayed a similar growth profile, survival capacity, and ability to infect feline, canine, and human monocyte-derived primary macrophages. Furthermore, multivariate cluster analysis suggested that most strains studied did not display distinctive phenotypic features. To our knowledge, this is the first study to analyse the phenotypic behaviour of feline L. infantum strains. This study brings new insights into the hypothetical role of cats as reservoir hosts of L. infantum since the parasites found in them are phenotypically identical to those of dogs and humans. However, further studies on the transmission dynamics should be encouraged to fully establish the status of cats in the maintenance of L. infantum foci. [ABSTRACT FROM AUTHOR]

Abstract: 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-γ, 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. [Copyright &y& Elsevier]