Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome.

UNLABELLED: : BACKGROUND: This study describes immunological responses, diagnostic features, follow up and treatment outcomes from seventeen dogs with papular dermatitis due to Leishmania infection diagnosed by cytology or real time-PCR. METHODS: Specific Leishmania humoral and cellular immune responses were evaluated by means of an immunofluorescence antibody test in all cases and a delayed-type hypersensitivity (DTH) reaction to leishmanin in eight cases. The extent of infection was studied in several tissues including blood, lymph node, conjunctival and oral swabs, by means of PCR, at the time of diagnosis and during follow-up. Culture was performed on nine dogs from cutaneous lesions and lymph node aspirates and molecular typing was carried out on isolates based on ITS-1, ITS-2 and Haspb gene sequencing analysis. RESULTS: Cytological and molecular results from fine needle aspirates of papules were diagnostic in 8 out of 13 (61.5%) cases and in 14 out of 15 dogs (93.3%), respectively. In all dogs, specific anti-Leishmania antibody levels were low or absent. Blood and lymph node PCRs and lymph node culture were negative in all dogs. Three out of the nine dogs (33%) were positive by culture from cutaneous lesions. The three isolates were identified as ITS type A, however, polymorphism was observed in the Haspb gene (PCR products of 626 bp, 962 bp and 371 bp). DTH response was positive in all tested dogs at the time of diagnosis. The majority of dogs were successfully treated with only N-methylglucamine antimoniate, after which cutaneous lesions disappeared or were reduced to depigmented, flattened scars. All dogs remained seronegative and the majority of dogs were negative by PCR in several tissues during follow-up. CONCLUSIONS: This study points out that papular dermatitis due to L. infantum is probably an underestimated benign cutaneous problem, associated with a parasite specific cell mediated immunity and a poor humoral immune response. Papular dermatitis is seen in young dogs, and appears to be a mild disease with restricted parasite dissemination and a good prognosis. PCR can be used as a non-invasive method to routinely evaluate papules if Leishmania infection is suspected in cases in which parasites are not visualized by cytology.

Prevalence of antibodies against Rickettsia conorii, Babesia canis, Ehrlichia canis, and Anaplasma phagocytophilum antigens in dogs from the Stretto di Messina area (Italy).

The aims of this study were to determine the seroprevalence for Rickettsia conorii, Ehrlichia canis, Anaplasma phagocytophilum, and Babesia canis in outdoor-kennelled dogs (n=249) from the Stretto di Messina (Italy) and to compare seroprevalence in 2 public shelters and 4 privately-owned kennels where different tick-preventive measures were implemented in order to focus on the specific sanitary risk posed by public shelters in southern Italy for tick-borne pathogens. R. conorii (72%) and B. canis (70%) were the most prevalent infections when compared to E. canis (46%) and A. phagocytophilum (38%). Seroprevalence for R. conorii, E. canis, and A. phagocytophilum was significantly higher in public shelters than in private kennels. However, B. canis seropositivity was similar in both types of kennels. In addition, in private kennels where a regular ectocide treatment was carried out by means of spot-on devices, dogs did not present E. canis and A. phagocytophilum antibodies. One hundred fifty-one dogs out of 249 (61%) were seropositive to more than one pathogen with R. conorii and B. canis the most common ones. Coinfections were more frequently found in public-shelter dogs. This study demonstrated high seroprevalences against R. conorii, B. canis, E. canis, and A. phagocytophilum in kennelled dogs from both coastal sites of the Stretto di Messina and the importance of regular tick-bite prevention by means of individual spot-on devices.

Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques.

The use of non invasive sampling, such as collection of conjunctival swabs, as a diagnostic tool for the detection of Leishmania DNA is of interest. The purpose of this study was to evaluate the diagnostic utility of detecting Leishmania infection with the use of conjunctival swab samples in dogs living in a highly endemic area for leishmaniosis and to investigate, for the first time, the presence of Leishmania DNA in oral swabs in the same population. One hundred sixty-three dogs living outdoor and recruited in various provinces of Sicily were studied. Leishmania infantum indirect fluorescent antibody test (IFAT), delayed-type hypersensitivity reaction to leishmanin (DTH) and real-time PCR of blood (BL), lymph node (LN), conjunctival (CS) and oral swab (OS) samples were performed. The positive PCR percentages in LN, CS, OS and BL samples were: 24.5%, 22.1%, 8.7% and 5.5%, respectively. Serological and DTH positive percentages were 27.0% and 73.8%, respectively. Seropositive and LN-PCR positive dogs had a high likelihood to be positive by CS-PCR. The similar positive PCR percentages found in CS and LN samples suggest the use of CS-PCR as non-invasive alternative technique to LN-PCR for the detection of Leishmania infection in dogs. In addition, this study demonstrated, for the first time, the presence of Leishmania DNA in oral swabs in dogs.

Efficacy of the fipronil 10%+(S)-methoprene 9% combination against Rhipicephalus sanguineus in naturally infested dogs: speed of kill, persistent efficacy on immature and adult stages and effect of water.

This field trial was designed to test the efficacy, in terms of treatment and prevention, of the fipronil 10%+(S)-methoprene 9% combination against immature and adult stages of Rhipicephalus sanguineus in naturally infested dogs, and to assess the effect of a single plain water exposure. Twenty-four dogs of various age, sex, weight and coat length were divided into two homogeneous groups, treated (T) and control (C), and housed into twin outdoor kennels. Trial baseline was designed as day 0, when dogs from group T were treated with a commercial spot-on formulation of fipronil 10%+(S)-methoprene 9%, while subjects from group C were left untreated and served as control. After treatment, tick load for each included dog was estimated, for both adult and immature ticks, using the localization and count over the entire body surface at the following time-points: day 2, to evaluate the speed of kill and at days 7, 14, 21 and 28 to assess the persistence of efficacy. The effect of water exposure on the product efficacy was tested at day 14 of the study, when six dogs, homogenously selected from group T, were soaked through with plain water. The overall tick load in dogs from group C was high throughout the entire study period, ranging from 103.2 (day 28) to 161.3 (day 0), and confirmed the high tick pressure. Speed of kill calculated at 48 h post-treatment was slightly higher for adult ticks (96.2%) than for immature stages (91.6%). Compared to the control, dogs treated with the fipronil+(S)-methoprene maintained a significantly lower mean tick load for both adult and immature stages in the four weeks of observation. Persistence of efficacy against immature stages ranged from 97.1% the first week, 99.6% second week, 99.7 third week and 93.1% in the last week. In the same way, efficacy against adult ticks was constantly high, shifting from 94.5% to 92.5%. Overall efficacy (adults+immatures) was the strongest in the first two weeks (i.e., 96.1% and 96.6%) and lowered in the two remaining weeks (i.e., 94.2% and 93.4%). Results showed that water exposure did not affect the activity and persistence of the tested combination. On the basis of this field trial, the fipronil+(S)-methoprene combination represents a highly efficacious product to control all stages of R. sanguineus ticks on dogs.

Prevalence and geographical distribution of canine hemotropic mycoplasma infections in Mediterranean countries and analysis of risk factors for infection.

Two hemoplasma species are known in dogs: Mycoplasma haemocanis (Mhc) and 'Candidatus Mycoplasma haematoparvum' (CMhp). Although their transmission routes are poorly understood, Rhipicephalus sanguineus has been suggested as a potential tick vector. The aim of the present study was to assess the prevalence, risk factors, and clinical importance of canine hemoplasmas in countries with a Mediterranean climate where R. sanguineus is highly prevalent using TaqMan real-time PCR, and to molecularly characterize the identified isolates. DNA (canine glyceraldehyde-3-phosphate dehydrogenase) was successfully amplified from all samples collected from 850 dogs in Italy, Spain, and Portugal, and 82 (9.6%) were PCR-positive for canine hemoplasmas (43 Mhc, 34 CMhp and 5 co-infected). The hemoplasma sample prevalence was significantly higher in Portugal (40%) than in Italy (9.5%) and Spain (2.5%). Risk factors for infection included living in kennels, young age, crossbreeding, and mange infection. No association was found with anemia. Phylogenetic analyses of the 16S rRNA and RNase P genes revealed >99% identity to other European isolates. In conclusion, canine hemoplasma infections were readily encountered in Mediterranean countries. The climate and living conditions seemed to influence canine hemoplasma prevalence. The clinical importance of canine hemoplasma infections appeared to be low, but the infection stage of the presented dogs was unknown.

Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection.

In this study, we demonstrate that the in vitro interactions between a CD56(neg)/CD16(pos) (CD56(neg)) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1-infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK-DC activation and maturation as well as a defect in the NK cell-mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56(neg) NK cell subset, largely accounts for the highly defective NK cell-mediated lysis of autologous iDCs. Moreover, mDCs generated from HIV-1 viremic but not aviremic patients are substantially impaired in their ability to secrete interleukin (IL)-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of interferon-gamma.

Identification of NKG2A and NKp80 as specific natural killer cell markers in rhesus and pigtailed monkeys.

Investigations of natural killer (NK) cells in simian models of disease have been hampered by a lack of appropriate phenotypic markers and by an inadequate understanding of the regulation of NK cell activities. In the present study, a panel of monoclonal antibodies (mAbs) specific for various human NK receptors was screened for cross-reactivity with NK cells from rhesus macaques and pigtailed macaques. Flow cytometric analyses using anti-human NKG2A and anti-human NKp80 mAbs individually, and particularly in combination with anti-CD16 mAb, allowed for the identification of the entire NK cell population in both species. NK cells in monkeys were generally identified by negative selection of peripheral blood mononuclear cells (PBMCs) for the absence of T-cell, B-cell, and monocyte markers. mAb-mediated ligation of NKp80 induced NK cell cytotoxicity, while in the case of NKG2A it displayed a clear capability to inhibit the lysis of target cells by NK cells from macaques, as well as from humans. This new phenotypic and functional characterization of NKG2A and NKp80 in rhesus and pigtailed macaque NK cells provides a new approach in the analysis of their innate immune system.

Characterization of CD56-/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals.

Natural killer (NK) cells are an important component of the innate immune response against viral infections. NK cell-mediated cytolytic activity is defective in HIV-infected individuals with high levels of viral replication. In the present study, we examined the phenotypic and functional characteristics of an unusual CD56(-)/CD16(+) (CD56(-)) NK subset that is greatly expanded in HIV-viremic individuals. The higher level of expression of inhibitory NK receptors and the lower level of expression of natural cytotoxicity receptors observed in the CD56(-) NK fraction compared with that of CD56(+) NK cells was associated with extremely poor in vitro cytotoxic function of this subset. In addition, the secretion of certain cytokines known to be important in initiating antiviral immune responses was markedly reduced in the CD56(-), as compared with the CD56(+) NK cell subset. These data suggest that the expansion of this highly dysfunctional CD56(-) NK cell subset in HIV-viremic individuals largely accounts for the impaired function of the total NK cell population.

Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates.

Natural killer (NK) cells play a central role in host defense against various pathogens. Functional defects of NK cells in HIV-1 infection as a direct effect of abnormal expression or function of inhibitory NK receptors (iNKRs), activating natural cytotoxicity receptors (NCRs), and NKG2D have not yet been described. This study demonstrates an expansion of the functionally defective CD56-/CD16+ population of NK cells in viremic versus aviremic patients. We also demonstrate that in HIV-infected viremic patients, expression of iNKRs was well conserved and that in most cases, there was a trend toward increased expression on NK cells as compared with healthy donors. It was also demonstrated that the major activating NK receptors, with the exception of NKG2D, were significantly down-regulated. In contrast, the expression of iNKRs and activating receptors in HIV-infected individuals whose viremia was suppressed to below detectable levels by highly active antiretroviral therapy for 2 years or longer was comparable to that of healthy donors. Functional tests confirmed that the abnormal expression of the activating receptors and of iNKRs was associated with a markedly impaired NK cytolytic function. This phenomenon is not attributed to a direct HIV-1 infection of NK cells; thus, this study may provide insight into the mechanisms of impaired host defenses in HIV-1 viremic patients.

Synthesis of new P3CS derivatives and their mitogenic activity on in vitro mice splenocytes.

Vaccination against tumors represents a relevant issue in current human cancer therapy. The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-Cys-Ser (P(3)CS) and analogs with longer aminoacidic sequence are polyclonal activators for B-lymphocytes. Previous study reported that their N-2,2,2-trichloroethoxycarbonyl (Troc) derivatives increase immunocyte mitogenic activity. Therefore, in order to obtain compounds of greater activity and to investigate relationships between molecular structure of S-glyceryl skeleton and biological activity, we synthesized new Troc derivatives of P(3)CS. The mitogenicity of compounds was determined in vitro, by measuring in vitro [3H]-thymidine incorporation into splenocytes from Balb/c mice. Concentrations of compounds ranged from 0 to 64 micro g/ml. In particular, S-[2,3-bis(trichloroethoxycarbonyloxy)]-N-trichloroethoxycarbonyl dipeptide derivative exhibited significant mitogenic activity endowed with high pharmacological potency. These new series of compounds could be used as potent immunoadjuvants for the development of novel synthetic vaccines for tumor immunotherapy.

Heme oxygenase-1 gene expression attenuates angiotensin II-mediated DNA damage in endothelial cells.

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression using a retroviral vector on attenuation of angiotensin II (Ang II)-mediated oxidative stress. Comet and glutathione (GSH) levels were used as indicators of the levels of oxidative stress. Comet assay was performed to evaluate damage on DNA, whereas GSH levels were measured to determine the unbalance of redox potential. Pretreatments with inducers, such as heme 10 microM, SnCl(2) 10 microM, and inhibitors, such as tin-mesoporphyrin 10 microM was followed by treatment with Ang II 200 ng/ml. Pretreatment with heme or SnCl(2) provoked significant reductions (P < 0.01) of tail moment in the comet assay. Opposite effects were evident by pretreatment for 16 hr with tin-mesoporphyrin. A decrease in tail moment levels was found in human endothelial cells transduced with the human HO-1 gene. The addition of Ang II (200 ng/ml) to human dermal microvessel endothelial cell-1 for 16 hr resulted in a significant (P < 0.05) reduction of GSH contents control endothelial cells but not in endothelial cells transduced with HO-1 gene. The results presented indicated that stimulation or overexpression of HO-1 attenuated DNA damages caused by exposures of Ang II.

Nerve growth factor-endothelial cell interaction leads to angiogenesis in vitro and in vivo.

Nerve growth factor (NGF) has important functions during embryonic development and on various tissues and organs under normal and pathological conditions during the extrauterine life. RT-PCR analysis and immunological methods demonstrate that human umbilical vein endothelial cells (HUVECs) express the NGF receptors trkA(NGFR) and p75NTR. NGF treatment caused a rapid phosphorylation of trkA(NGFR) in HUVECs, determining a parallel increase of phosphorylated ERK1/2. Accordingly, NGF induced a significant increase in HUVEC proliferation that was abolished by the trkA(NGFR) inhibitor K252a. Also, HUVECs express significant levels of NGF under standard culture conditions that were up-regulated during serum starvation. Endogenous NGF was responsible for the basal levels of trkA(NGFR) and ERK1/2 phosphorylation observed in untreated HUVEC cultures. Finally, NGF exerted a potent, direct, angiogenic activity in vivo when delivered onto the chorioallantoic membrane of the chicken embryo. The data indicate that NGF may play an important role in blood vessel formation in the nervous system and in several pathological processes, including tumors and inflammatory diseases. Unraveling mechanisms of NGF-dependent angiogenesis could provide valuable tools for novel therapeutic approaches in antiangiogenic therapy.