Abstract: In this study, 19 chickens were experimentally infected by Leishmania infantum and tissue samples, collected at different times, were cultured and subjected to conventional PCR and/or real time PCR (qPCR) to assess their susceptibility to infection. In addition, 121 serum samples from rural chickens (n=73) and backyard birds (n=48) were tested for anti-L. infantum antibodies by indirect immunofluorescence test. All the 19 animals showed to be molecularly positive at least at one tissue sample. In particular, 26 tissue samples from the experimentally infected chickens were positive on conventional PCR and/or qPCR but no clinical signs or seroconversion were detected and all tissue cultures were negative. Accordingly, all serum samples from rural chickens were negative whereas four (8.4%) from game birds (three Anser anser and one Phasianus colchicus) were positive. These results indicate that chickens are not suitable hosts for L. infantum under experimental condition. The occurrence of anti-L. infantum antibodies in domestic gooses (A. anser) and in a pheasant (P. colchicus) points out their possible role in the epidemiology of visceral leishmaniasis.
Abstract: The aim of this study was to evaluate, in terms of dog macrophage killing ability in vitro, a vaccine based on Leishmania infantum promastigote soluble antigen (LSA) formulated with three different adjuvants (BCG, AdjuPrime, MPL/TDM/CWS). A significant increase of the macrophage killing ability was observed in dogs vaccinated with LSA+MPL/TDM/CWS after 1 month from vaccination. A similar increase of macrophage parasitocidal ability was present only after 5 months in dogs vaccinated with LSA+BCG or LSA+AdjuPrime. In all dogs the augmented killing percentage was still present after 12 months from vaccination. Therefore, in particular LSA+MPL/TDM/CWS vaccine seems promising for further studies in dogs.
Abstract: The aim of this study was to evaluate cytokine expression in 22 Leishmania infantum naturally infected dogs, in order to correlate this parameter with the clinical status of infected animals. After 4 and 8 months from the first diagnosis of Leishmania infection, clinical and laboratory examination of dogs was performed and peripheral blood mononuclear cells (PBMC) were isolated. The cytokine profile was analysed in terms of IFN-gamma, IL-4, IL-10 and TNF-alpha mRNA expression in cultured PBMC by a semi-quantitative reverse transcriptase-PCR. Thirteen out of 22 Leishmania-infected dogs remained asymptomatic in the follow-up, while 9 showed clinical signs of leishmaniasis. IL-4, IL-10, TNF-alpha and IFN-gamma mRNA levels were not significantly different in asymptomatic compared to symptomatic animals 4 months from the diagnosis of Leishmania infection, but were significantly higher in symptomatic versus asymptomatic dogs after 8 months from diagnosis. In addition, IL-4, IL-10 and TNF-alpha mRNA levels significantly increased only in symptomatic dogs at 8 months, in comparison to the levels found at 4 months. These results show a mixed Th1 and Th2 cytokine response in Leishmania-infected dogs, with higher cytokine expression in dogs with manifest clinical disease, during the second follow-up after 8 months from the first diagnosis of infection.
Abstract: Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.
Abstract: ABSTRACT: BACKGROUND: Human and canine leishmaniasis (CanL) by Leishmania infantum is endemic in Italy, with a high percentage of infected asymptomatic animals. However, the immune response mechanisms underlying the clinical presentation of CanL have not been fully investigated. Among leishmanicidal molecules produced by activated macrophages, nitric oxide (NO) produced by an inducible NO synthase seems to play an important protective role, but no conclusive data are available. Therefore, NO released by cultured macrophages from dogs with natural Leishmania infection living in an endemic area for CanL was evaluated. METHODS: On the basis of one year's clinical and laboratory follow-up, 22 dogs infected by Leishmania infantum were identified and grouped as: asymptomatic dogs (n = 13) and dogs with symptoms of leishmaniasis (n = 9). Each animal was bled twice at 4-month intervals and macrophage and lymphocyte cultures were obtained from peripheral blood mononuclear cells. Supernatants of L. infantum-infected macrophage cultures, with or without addition of autologous lymphocytes, were assayed for NO production by Griess reaction for nitrites. RESULTS: In the first months of the infection the levels of NO in supernatants of Leishmania-infected macrophages were higher in symptomatic than in asymptomatic dogs, but they were significantly increased in the latter group eight months after the diagnosis of infection. Furthermore, NO release significantly decreased in the presence of autologous lymphocytes in both groups of animals. CONCLUSION: These results suggest that NO may be involved in the long-term protection of dogs against natural Leishmania infection and in the clinical presentation of canine leishmaniasis in the Mediterranean area.
Abstract: Giardia and Cryptosporidium spp. are protozoa that cause human and animal disease worldwide and often exhibit zoonotic transmission. This review gives ample information concerning the epidemiology of these parasites in Italy, i.e. prevalence data in humans, farm and pet animals, shellfish and aquatic environment. Moreover, it reports genotyping results obtained from different isolates, with particular emphasis on the spread of host-specific and zoonotic species/genotypes of various origin, and on molecular data that make the Italian situation different from that of other countries. Finally, possible explanations are given for the infrequent reports of Giardia and Cryptosporidium spp. outbreaks, despite widespread faecal contamination by these parasites.
Abstract: Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.
Abstract: Anisakidosis is a parasitic disease of the human gastrointestinal tract caused by ingestion of larvae of marine nematodes such as Anisakis spp. or, rarely, Pseudoterranova spp., present in raw or undercooked fish. We report the first series of gastric Anisakis infection (anisakiasis) from a single centre in Italy. In our department, we observed 3 cases, all in women who were urgently hospitalized following intense epigastric pain and vomiting, developed after the ingestion of raw fish. The patients underwent urgent gastroscopy within a few hours. In each, a worm was extracted from the gastric mucosa by means of biopsy forceps. This was followed by prompt clinical improvement. The worm was identified by its macroscopic and microscopic characteristics as an Anisakis spp. larva (L3). In 2 cases, laboratory tests revealed marked leukocytosis and eosinophilia in the peripheral blood 3-4 days after ingestion of the raw fish. The diagnosis of anisakiasis can be made by endoscopy, radiology and abdominal ultrasound, but is often made only at surgery. In the gastric form of the disease, urgent gastroscopy has both a diagnostic and a therapeutic role, because the worm can be removed by means of biopsy forceps.
Abstract: Giardia and Cryptosporidium spp. are parasitic protozoa which are frequent etiologic agents of waterborne diseases. This lecture will summarize the main biological and environmental factors involved in the potential risk for waterborne transmission of giardiosis and cryptosporidiosis, which have caused many outbreaks in different geographical areas. In particular, the current epidemiological situation of these parasitoses in Italy will be analysed, on the basis of research carried out on humans and on the environment. Finally, current methods for evaluating the presence of Giardia cysts and Cryptosporidium oocysts in water and new methods for cyst/oocyst removal from drinking water and wastewater will be examined.
Abstract: A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.
Abstract: The purpose of this study was to evaluate the prevalence of intestinal parasites in 277 healthy subjects in the city of Mamuras (Albania, South Eastern Europe) and the correlation between parasitic infections and possible risk factors. Faecal samples collected with sodium-acetate-formalin fixative were concentrated by formalin ethylacetate sedimentation and examined as wet mounts, permanent stains and by anti-Giardia/Cryptosporidium fluorescent antibodies. Data concerning age, sex, level of education, availability of piped water, number of people living in the same house, and residence in rural or urban area were collected for each subject. Statistical analysis was performed by chi-square test and regression logistic analysis. The overall prevalence of intestinal parasites was 183/277 (66.06%). In particular, pathogenic protozoa or helminths were found in 67 subjects (24.18%), including Trichuris trichiura in 34 (12.27%), Giardia duodenalis in 31 (11.19%), Hymenolepis nana in 5 (1.8%), Ascaris lumbricoides in 3 (1.08%). A significant correlation was observed only between parasite colonization and older age and between Trichuris trichiura infection and residence in rural areas.
Abstract: Dendritic cells (DCs), instructed by the priming signals from microbial factors, can produce interleukin (IL)-12p70 and promote T helper (Th)1 proliferation and interferon (IFN)-gamma production. This event seems to be critical for the containment of infections caused by intracellular pathogens, even including Leishmania infection. In the present in vitro study we have investigated: 1) phagocytic capacities and IL-12 production by human monocyte-derived DCs and macrophages (MØs), infected with Leishmania infantum promastigotes; 2) IFN-gamma production by human CD4+ T cells co-incubated with DCs or macrophages pulsed with live promastigotes. Monocyte-derived myeloid DCs and MØs from healthy donors were infected with live metacyclic Leishmania infantum (MON-1) promastigotes, previously opsonized with 5% autologous serum, at 1:4 cell/parasite ratio. Percentage and index of phagocytosis were calculated after 2, 24 and 48 h of incubation. IL-12 production was evaluated by an ELISA in supernatants from 48 h Leishmania-infected or lipopolysaccharides (LPS)-stimulated DCs and MØs, also in the presence of phytohemagglutinin-activated or inactivated CD4+ T cells. For IFN-gamma production, CD4+ T cells were repeatedly stimulated with DCs or MØs, pulsed with live Leishmania promastigotes or activated with LPS. The number of IFN-gamma-secreting cells was evaluated by an ELISpot assay. Results showed that MØs have a higher phagocytic capacity towards L. infantum promastigotes than DCs. Moreover, unlike MØs, Leishmania-infected DCs were able to release IL-12p70; this production significantly increased in the presence of activated CD4+ T cells. Finally, DCs pulsed with live parasites and added to autologous CD4+ T cells induced a higher number of IFN-gamma-secreting cells than MØs, thus indicating their ability to polarize Th cells toward the Th1 subset. These data indicate that DCs are able to promote protective Th1 immune responses in our experimental model of Leishmania infantum infection, thus representing the grounds for initiating immunoterapeutic and vaccinal strategies.
Abstract: The spread of human leishmaniasis has prompted the scientific community to study dogs as reservoirs for Leishmania infantum. Canine leishmaniasis (CanL) is widespread in the Mediterranean area with a prevalence of up to 50%. The first step toward controlling the disease is to monitor its distribution, mainly in stray dogs. The validity of a recombinant K39 (rK39) dipstick test, commercially available for the serodiagnosis of human leishmaniasis, was evaluated using sera from 165 dogs selected on the basis of positive or negative lymph node smears at parasitological examination. The results were compared with the indirect fluorescent antibody test (IFAT) (cutoff 1:80). Sera from a group of dogs with other diagnosed diseases but negative for leishmaniasis were also tested to evaluate any cross-reactivity. Various procedures were used for testing whole blood samples. The relative specificity of the rK39 dipstick and IFAT was 100% (97 of 97) and 98.97% (96 of 97), whereas the relative sensitivity was 97.06% (66 of 68) and 98.53% (67 of 68), respectively. The results of the dipstick and IFAT corresponded except for 2 sera (k = 0.987). This data confirm the usefulness of rK39 antigen for diagnosing CanL both in symptomatic and asymptomatic dogs. The rK39 dipstick proved to be a rapid, sensitive, and specific test that may be very useful in the field for large-scale screening and also in veterinary practice, requiring minimal equipment and operator expertise.
Abstract: Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D-induced apoptosis of the human monocytic cell line U-937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite-free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis-inducing agent. Actinomycin D-induced apoptosis in U-937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U-937 cells via a mechanism involving inhibition of caspase-3 activation. Furthermore, protein kinase C delta (PKC delta) cleavage was increased in actinomycin D-treated U-937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC-mediated pathways by LPG can be implicated in the enhanced survival of the parasites. These results support the claim that promastigotes of L. infantum, as well as its surface molecule, LPG, which is in part released in the culture medium, inhibit macrophage apoptosis, thus allowing intracellular parasite survival and replication.
Abstract: An rK39 immunochromatographic test and immunofluorescent-antibody test (IFAT) for serodiagnosis of canine leishmaniasis were evaluated. The two tests showed correlation for all but one of the sera obtained from 68 dogs confirmed as leishmaniasis cases and 40 dogs (22 healthy dogs and 18 dogs with other diseases) from areas where the disease is not endemic. Specificity was 100% for both tests, while sensitivity was 97% for the rapid test and 99% for IFAT.
Abstract: Chemokines exert their actions through G-proteinlinked receptors, which are expressed to variable extents by different cell types. In accordance with the chemokine classification, these receptors are designated as CXC, CC, XC, and CX(3)C, followed by R and a number. The purpose of this investigation was to evaluate CCR1 expression in human peripheral blood-derived macrophages and the human monocytic U-937 cell line. Cells in vitro were infected with live Leishmania infantum promastigotes (zymodeme MON1); cell lysates were then subjected to SDS-PAGE and immunoblotting, by using an anti-CCR1 affinity purified polyclonal antibody. The expression of the CCR1 gene was analyzed by RT-PCR, using specific human primers. The results of both immunoblotting and RT-PCR showed that CCR1 expression in Leishmania-infected cells was lower than in uninfected control cells. These results indicate that Leishmania infantum infection causes a down-regulation of the CCR1 gene and protein expression, suggesting that reduced phagocyte recruitment at the inflammation sites could favor parasite progression and the spread of Leishmania infection.
Abstract: Leishmania is a parasitic protozoan which causes severe disease in humans and dogs. Screening of new compounds against leishmaniasis is particularly needed to determine the toxicity of some existing drugs and the appearance of drug-resistant strains. Reliable and simple in vitro models are required for large-scale initial screenings. In this review different methods for in vitro evaluation of drug activity against Leishmania spp. are summarized.
Abstract: Dendritic cells (DCs) are key elements of the immune system, which function as sentinel in the periphery and alert T lymphocytes about the type of invading antigen and address their polarisation, in order to mount an efficacious immune response. Leishmania spp. are parasitic protozoa which may cause severe disease in humans and domestic animals. In this work, the main studies concerning the role of DCs in Leishmania infection are reviewed, in both the murine and human models. In particular, the importance of the genetic status of the hosts and of the different Leishmania species in modulating DC-mediated immune response is examined. In addition, different approaches of DC-based vaccination against experimental leishmaniasis, which could have important future applications, are summarised.
Abstract: Nitric oxide (NO) is a pleiotropic mediator of numerous biological processes, including smooth muscle relaxation, neurotransmission and defence against pathogens. In addition, NO is involved in the pathogenesis and control of inflammation, tumors, autoimmunity, and infectious and chronic degenerative diseases. NO, a highly reactive radical, is produced from L-arginine and oxygen by the enzyme NO synthase (NOS). Three NOS isoforms have been identified: two distinct NOS isoforms are constitutively expressed in cells, whereas a third isoform, inducible NOS (iNOS), is transcribed in response to specific stimuli. In particular, iNOS is responsible for the discontinuous synthesis of high amounts of NO and was originally characterized in murine macrophages after exposure to cytokines and/or microbial products. A wide range of microorganisms is sensibly inhibited in its development by NO, like fungi, bacteria, protozoa and viruses. Although NO production and its antimicrobial effect appear well established in rodent macrophages, the existence of L-arginine pathway in human mononuclear phagocytes has long been disputed. Recently, evidences showing the iNOS activity and NO production in other animal models, including humans, are now emerging, even if the NO induction has been more difficult to demonstrate. The present observations provide evidence for the occurrence of iNOS protein expression and NO production in human macrophages cultured in vitro.
Abstract: Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1 alpha on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1 alpha significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-gamma. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L- N(G)monomethylarginine ( L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1 alpha mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.
Abstract: The purpose of this study was to compare the performance of a rapid immunochromatographic dipstick test for the qualitative detection of circulating antibodies to the leishmanial recombinant antigen K39 with that of a classical immunofluorescent antibody test for serodiagnosis of visceral leishmaniasis. Sera from 143 Italian subjects, including 69 patients with clinically suspected visceral leishmaniasis, 23 patients with hypergammaglobulinemia and 51 healthy controls, were tested. The immunochromatographic test was performed according to the manufacturer's instructions, using antigen-impregnated nitrocellulose paper strips. The immunofluorescent antibody test was performed according to an established method, using promastigotes of Leishmania infantum zymodeme Montpellier 1 as antigen. In 11 patients, diagnosis of active Leishmania infection was established by microscopic examination of biopsy samples and/or clinical response to meglumine antimoniate. Results of the two tests correlated for all but two sera examined. In two patients, one with proven infectious mononucleosis and one with bacterial pneumonia, the immunofluorescent antibody test was positive and the dipstick test was negative. In the restricted sample of patients in whom a definitive diagnosis was established, the immunochromatographic test was positive in 11 of 11 patients with confirmed Leishmania infection and negative in 103 of 103 subjects who either had other documented diseases or were healthy controls, showing 100% sensitivity and 100% specificity.
Abstract: An unusual presentation of leishmaniasis that occurred in an Italian immunocompetent woman is described. The patient had a long history of coagulopathy due to factor VIII deficiency and pain in the right lumbar region. Computed axial tomography demonstrated a cystic mass in the right adrenal gland. Histological examination of the surgically removed cyst showed the presence of histiocytes containing Leishmania amastigotes. Serodiagnosis for leishmaniasis performed through immunofluorescent antibody testing and the rK39 enzyme immunoassay was positive, whereas a bone marrow aspirate did not reveal any parasite. The patient was not treated for leishmaniasis and recovered well after surgery. Serological testing was still positive 2 years after surgery, but clinical follow-up did not reveal the signs typical of visceral leishmaniasis.
Abstract: Human visceral leishmaniosis is endemic in Southern Italy, where the dog is the main reservoir of viscerotropic strains of Leishmania infantum. The release of nitric oxide (NO) by interferon (IFN)-gamma-activated macrophages is an important leishmanicidal mechanism in several animal species. In this work NO production, phagocytosis and killing capacity of monocyte-derived dog macrophages were evaluated in vitro before and after administration of a vaccine composed of killed Leishmania infantum promastigotes. Moreover, IFN-gamma content was measured in concanavalin A-activated dog peripheral blood mononuclear cell (PBMC) supernatants employed for macrophage stimulation. Phagocytosis, killing capacity and NO production by canine macrophages increased significantly 1 month after vaccine administration, and the increase also persisted 5 months later. In addition, the amount of IFN-gamma in PBMC supernatants was significantly higher after vaccination. Overall, our results suggest the usefulness of evaluating the in vivo protective role of this promastigote preparation in dogs.
Abstract: Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS or NOS2) plays a major microbicidal role in murine macrophages and its importance is now emerging also in the dog and human models. In dogs we demonstrated that macrophages in vitro infected with Leishmania infantum produced NO, after stimulation with cytokine-enriched peripheral blood mononuclear cell supernatants. In addition, parasite killing was reduced by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine blood monocytes before macrophage differentiation did not release NO, and their leishmanicidal activity was instead correlated with superoxide anion and interferon (IFN)-gamma production. In human macrophage cultures, after infection with Leishmania infantum, we showed both iNOS expression by immunofluorescence and western blotting and NO release by the Griess reaction for nitrites. Various cytokines and prostaglandins can differently modulate NO synthesis. In our experiments, stimulation by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly enhanced iNOS expression and NO production in human macrophages. In addition, the prostaglandin E2 increased NO release in activated, Leishmania-infected human macrophages. These results are interesting in the light of a possible immunological or pharmacological regulation of NO synthesis and microbicidal functions of macrophages.
Abstract: Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-gamma and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E2 has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E2. Experiments were also performed in the presence of the nitric oxide synthase inhibitor L-NGmonomethylarginine (L-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E2 exhibited increased nitric oxide production and parasite killing, which were significantly reduced by either L-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E2. Taken together, these results indicate that prostaglandin E2 may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E2 on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E2 synthesis.
Abstract: Nitric oxide (NO) production by the inducible NO synthase (iNOS or NOS2) represents one of the main microbicidal mechanisms of murine macrophages, but its role in other animal models is poorly investigated. Therefore, the aim of this work was to evaluate NOS2 expression in dog macrophages infected with Leishmania infantum. Macrophages obtained from peripheral blood of healthy dogs were activated with recombinant human interferon (rhIFN)-gamma and bacterial lipopolysaccharide (LPS) and then infected with L. infantum promastigotes. zymodeme MONI. For the immunofluorescence assay fixed macrophages were incubated with polyclonal rabbit anti-NOS2 and then with rhodamine F(ab')2 goat anti-rabbit IgG. For immunoblotting, cell lysates were submitted to SDS-PAGE and blots were incubated with polyclonal rabbit anti-NOS2 and then with horseradish peroxidase-conjugated goat anti-rabbit IgG. Results demonstrated that L. infantum-infected cells, after stimulation with rhIFN-gamma and LPS, displayed high levels of fluorescence for the NOS2 in their cytoplasm, unlike unstimulated uninfected macrophages. In western blotting, polyclonal anti-NOS2 reacted specifically with a protein band corresponding to 130 kDa. The signal produced in Leishmania-infected cells stimulated with rhIFN-gamma and LPS was higher than that produced in Leishmania-infected unstimulated cells. No band was detected in cellular lysates from uninfected unstimulated cells. These results indicate that dog macrophages can express NOS2, and suggest a role for IFN-gamma and LPS in NOS2 induction also in this animal model.
Abstract: Several species of protozoa belonging to the genus Leishmania are pathogenic for humans, causing visceral and cutaneous diseases. They are transmitted by phlebotomine sandflies as flagellated promastigotes to mammals hosts, where they live as aflagellated amastigotes mainly within macrophages. Studies performed on mice infected with Leishmania major demonstrated that host defence against this infection depends on the interleukin-12-driven expansion of the T helper 1 cell subset, with production of cytokines such as interferon-gamma, which activate macrophages for parasite killing through the release of nitric oxide. The parasitocidal role of this radical is now emerging also in the human and canine model. Healing or progression of the infection is related to the genetic and immune status of the host, and to the virulence of different species and strains of Leishmania. The parasite survival ultimately depends on the ability to evade the host immune response by several mechanisms. Among them, inhibition of the signal transduction pathway of the host cells is particularly important. In fact, promastigotes inhibit protein kinase C activation, cause Ca++ influx into the host cell and decrease the levels of myristoylated alanine-rich C kinase substrate-related proteins, which are substrates for PKC. In addition, Leishmania infection blocks IFN-gamma-induced tyrosine kinase phosphorylation, with consequent impairment of signalling for IL-12 and nitric oxide production. Finally, Leishmania activates protein phosphotyrosine phosphatases, which down-regulate mitogen-activated protein kinase signalling and c-fos and nitric oxide synthase expression. New pharmacological applications, including protein tyrosine phosphatase and protein farnesyltransferase inhibitors, are being evaluated against leishmaniosis in vitro and in vivo in the murine model.
Abstract: Giardia and Cryptosporidium are important agents of water-borne parasitic diseases. In this work we have examined the recovery efficiency of two methods for concentrating Giardia cysts and Cryptosporidium oocysts from water: a membrane filtration method and a crossflow filtration method. Results demonstrated a higher recovery efficiency for crossflow filtration method in comparison to the membrane filtration method. In addition, Giardia cysts and Cryptosporidium oocysts concentration was evaluated in wastewater samples submitted to chemical flocculation or chemical flocculation followed by slow sand filtration. Results showed that slow sand filtration was capable of reducing the number of Giardia cysts, but not of Cryptosporidium oocysts in wastewater.
Abstract: To better understand whether potent antiretroviral therapies can modify the natural history of HIV-1-associated microsporidiosis and cryptosporidiosis, the response to antimicrobial treatment of these opportunistic infections was evaluated in patients with or without antiretroviral treatment. Fifty patients with diarrhoea, all positive for Cryptosporidium parvum or Enterocytozoon bieneusi, were included in the study. Retrospective data were collected concerning demographics, clinical and microbiological characteristics of the parasitic infection, antiretroviral therapy and prophylaxis against opportunistic infections. Faecal samples were prepared using the Richie formalin-ethyl acetate method and stained using the modified Ziehl-Neelsen method for detection of Cryptosporidium parvum and Isospora belli, the modified trichrome and calcofluor white technique for detection of Enterocytozoon spp., and iodine for detection of ova, cysts or vegetative forms. Diarrhoea was defined as an abnormal increase in stool liquidity, an abnormal increase in stool frequency and a daily stool weight of more than 250 g for a period of at least 4 days. Patients treated with double antiretroviral therapy or protease inhibitors demonstrated an excellent response and a sustained therapeutic effect after follow-up (range, 5-36 months). The relapse of cryptosporidiosis in two patients who discontinued antiretroviral therapy suggests that the infection might remain in a latent stage. The resolution of the diarrhoea seems to be related to an increased CD4+ cell count rather than to the viral load. In conclusion, these data strongly support the hypothesis that combination antiretroviral therapy is able to greatly modify the course of cryptosporidiosis and microsporidiosis in patients infected with HIV-1.
Abstract: Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.
Abstract: A set of genetic markers, based on PCR-RFLPs of three diagnostic restriction enzymes (Hhal, Hinfl and Taql), which proved to be suitable for the identification of the species of the genus Anisakis, was used for the first molecular identification of a larva obtained by endoscopy in a case of gastric anisakidosis, in a 51 year old woman from Southern Italy. The analysis of the restriction profiles obtained allowed the larva to be identified as Anisakis pegreffii, one of the three sibling species of the A. simplex complex. PCR-RFLP proved to be a cost-effective and reliable tool for the exact identification of Anisakis larvae recovered from infected humans.
Abstract: Protozoa are important enteric pathogens in patients with human immunodeficiency virus (HIV) infection. In this study the prevalence of intestinal protozoa in 154 HIV-infected patients, with or without diarrhoea, in our region (Apulia, South Italy) was evaluated between December 1993 and February 1998. In the majority of patients CD4+ T cell count was below 200/microl. The overall prevalence of intestinal protozoa was 43/154 (27.92%). Twenty-eight (43.08%) out of 65 patients with diarrhoea and 15 (16-85%) out of 89 non-diarrhoeic patients were parasitized. In particular, in the group of 65 patients with diarrhoea the following protozoa were identified: Cryptosporidium parvum in 14 (21.54%), Blastocystis hominis in 7 (10.77%), microsporidia in 6 (9.23%), Giardia lamblia in 4 (6.15%) and Isospora belli in 1 (1.54%). Three patients were Cryptosporidium parvum-microsporidia co-infected. In patients without intestinal symptoms, prevalence was 3/89 (3.37%) for Cryptosporidium parvum, 9/89 (10.11%) for Blastocystis hominis, 1/89 (1.12%) for microsporidia and 2/89 (2.25%) for Giardia lamblia. A significant (P<0.001) correlation was observed between protozoan infection and the presence of diarrhoea. In particular, Cryptosporidium parvum and microsporidia infections were significantly (P<0.001) and P = 0.046, respectively) associated with diarrhoeal illness. Moreover, the majority of cases of cryptosporidiosis were first diagnosed in the periods of heaviest rainfall. Therefore, drinking water contamination may be a possible source of human infection in our area.
Abstract: Protozoa of the genus Leishmania infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. The parasitocidal mechanism of peripheral blood monocytes towards Leishmania in the dog has not been investigated in detail. Consequently, Leishmania infantum-infected monocyte cultures of healthy dogs were evaluated using the following parameters: (1) phagocytosis and killing capacities; (2) oxidative burst, in terms of superoxide anion (O2-) release, and (3) nitric oxide (NO) activity, in terms of nitrite (NO2-) production in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (NGMMLA). Parallel experiments were performed on monocytes stimulated with supernatants of concanavalin A-activated PBMC and on unstimulated monocytes. The amount of IFN-gamma in PBMC supernatants used for monocyte activation was determined by a biological assay on a canine Madin Darby cell line. Results demonstrated that phagocytosis, killing capacity and O2- production significantly increased in monocytes stimulated with supernatants, in comparison with unstimulated cells. A positive correlation was observed between the killing capacity, the O2- production and the amount of IFN-gamma in PBMC supernatants employed for monocyte activation. No significant differences were observed in NO production between unstimulated and stimulated cultures, or between the same cultures with and without NGMMLA. Finally, the killing percentage was similar in the presence or absence of NGMMLA, suggesting that in this experimental model peripheral blood dog monocytes lack NO-mediated killing.
Abstract: Protozoa of the genus Leishmania (L.) infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. Since the parasitocidal mechanism of macrophages towards Leishmania in dog has not yet been well investigated, in this work we have evaluated in Leishmania infantum-infected macrophage cultures from 10 healthy dogs, killing capacity and nitric oxide (NO) production, in terms of nitrite (NO2) levels. Parallel experiments were performed on macrophages stimulated with both Concanavalin A (ConA)-activated PBMC supernatants and Salmonella typhimurium lipopolysaccharide (LPS), and in the same conditions, but in the presence of the NO synthase inhibitor L-N monomethylarginine (L-NMMA). In L. infantum-infected macrophages, nitric oxide production was observed at a concentration significantly higher after stimulation with both Con A-activated PBMC supernatants and LPS than that observed in uninfected cells cultured in medium alone, or infected cells unstimulated or stimulated by PBMC supernatants or LPS alone, respectively. Moreover, NO production was abolished in the presence of the NO synthase inhibitor L-NMMA. Finally, killing of Leishmania by macrophages was significantly reduced in the presence of L-NMMA.
Abstract: We report the epidemiological, clinical and therapeutic characteristics of giardiasis in a population of HIV-infected patients with diarrhoic syndrome. During the period between 1988 and 1995, 720 HIV-patients with diarrhoic syndrome were evaluated. Fecal specimens were submitted to parasitological examination according to the Ritchie formalin-ethil acetate centrifugal sedimentation method and stained with iodine. Samples also underwent modified Ziehl-Neelsen staining and standard bacteriologic testing. Cystis of G. intestinalis were identified in stool sample of 25 patients. Two patients were classified as in stage A2 and 23 in C3. Mean CD4 values of patients with giardiasis (26.9 cells/mmc) were compared with those of 65 patients from whom, during the study, was isolated Cryptosporidium parvum (63.12, cells/mmc): the difference resulted highly significant (p < 0.001). Among the patients with full-blown AIDS, giardiasis occurred following a single previous AIDS-defining event in 13 inividuals, in seven and in five subjects giardiasis was the 3rd and, respectively, the 4th relevant AIDS-defining condition. Death occurred within the following 2 months in nine patients and within 6, 12 and 24 months in seven, six and two patients, respectively; at present only three AIDS patients are still alive. In general, G. intestinalis in HIV+, is not considered a major cause of enteritis; nevertheless, in our experience enteritis due to G intestinalis is a frequent event among AIDS patients, especially in the most advanced stage of disease, irrespectively of the risk factor. The increase in mean survival of AIDS patients will probably lead to a progressive emergence of this pathogen which could determine a severe diarroic syndrome with hydro-electrolytic impairments.
Abstract: Leishmania infantum is the causative agent of canine leishmaniasis in the Mediterranean area. The aspects of the canine immune response which may explain the chronic severe disease in these animals have not been well investigated. Therefore, in this work we have evaluated the parasite killing ability by circulating polymorphonuclear (PMN) cells and monocytes in dogs with spontaneous leishmaniasis and in healthy dogs. Moreover, in order to analyse the mechanisms responsible for killing activity, we evaluated the oxidative burst of circulating PMN cells, in terms of O2- and H2O2 production. The killing ability of infected dog PMN cells and monocytes was significantly lower than in a group of healthy dogs and a group of infected dogs treated with meglumine antimoniate and without symptoms of leishmaniasis. Superoxide anion and hydrogen peroxide production by PMN cells was significantly lower in a group of Leishmania-infected dogs than in healthy dogs, suggesting that these mediators may be involved in the reduced killing activity of infected dogs.
Abstract: Lipophosphoglycan (LPG) is the major glycoconjugate of Leishmania promastigote surface membrane. Previous studies on human and murine models have demonstrated that this molecule is involved in the attachment and survival of Leishmania in the host cells. Dog is the main reservoir of Leishmania strains responsible for human leishmaniasis in Italy. Since no studies have been performed on the LPG-canine phagocyte interactions, we investigated the LPG effects on dog phagocyte functions by evaluating: 1) the chemotactic activity of peripheral monocytes and polymorphonuclear (PMN) cells, in terms of cellular polarization; 2) the PMN cell respiratory burst, by measuring superoxide anion and hydrogen peroxide production. Results demonstrated a significant reduction of metabolic and chemotactic activity in LPG-preincubated cells, thus emphasizing the ability of this molecule to impair also the canine phagocyte responses.
Abstract: Cryptosporidium parvum is a protozoan which causes self-limiting diarrhea in immunocompetent subjects, and severe life-threatening disease in immunocompromised patients. Cryptosporidiosis is more common in developing countries and in infants. In this paper we have evaluated the prevalence of C. parvum in 368 hospitalized children with enteritis, of whom 359 were immunocompetent and 9 HIV-infected. Stool specimens were concentrated by sedimentation and stained with a modified Ziehl-Neelsen method. Cryptosporidium parvum oocysts were found in 7 (1.90%) out of 368 subjects. Six of these were immunocompetent (with an infection rate in this population of 1.67%) and 1 HIV-infected, asymptomatic except for diarrhea. In all children symptoms of enteritis and oocyst excretion cleared within 10 days. These results indicate that the prevalence of C. parvum as a causative agent of diarrheal illness in hospitalized immunocompetent children is rather low in our region (Apulia, South Italy).
Abstract: In this review we have summarized the main data concerning Leishmania-macrophage interactions, with particular emphasis on receptors involved in adhesion, activating or deactivating cytokines and toxic molecules responsible for parasite killing. At present it is also known that a different T helper (Th)1- or Th2-cell response may be critical for the outcome of Leishmania infection in human and in murine models. Therefore, we have mentioned the recent studies on cytokines, such as IL-2, which are able to cause the switch from a Th2, disease-promoting immune response, to a Th1, protective response. In fact, in the light of these findings, these molecules may be used in the future for immunotherapeutical or immunoprophylactic purposes.
Abstract: The present study reports the clinical features of two AIDS patients infected by Cyclospora. According to our observations, Cyclospora in AIDS can be responsible both for gastroenteritis and for asymptomatic infections with spontaneous rapid clearance. In addition, and undiagnosed circulation of this agent in Italy could be hypothesized: neither patient had a past history of foreign travel. These are the first two cases in AIDS described in Italy.
Abstract: Protozoan parasites of the Leishmania genus are the causative agents of important diseases in humans and animals. During their life cycle in vertebrate hosts, protozoa are able to live and proliferate within phagolysosomes of host phagocytic cells. The capacity to live in this hostile environment is likely due to the cell surface glycoconjugate expression. In particular, lipophosphoglycan (LPG), a major surface glycoconjugate of Leishmania promastigotes, has been reported to play an active role in protecting parasites within phagolysosomes via the impairment of killing mechanisms. In this review, the authors emphasize some novel LPG-mediated escape mechanisms of promastigotes from human phagocyte responses, such as the impairment of oxidative burst and of chemotactic activity. In the light of these findings, the knowledge of biological actions of LPG may be useful in order to prepare a vaccine against human leishmaniasis, using LPG defective avirulent mutant strains.
Abstract: Microsporidia are intracellular parasitic protozoa very common in immunocompromised patients in many parts of the world. There is a scarcity of data on the prevalence of these parasites in Italy. In this study we examined stool samples of 56 HIV+ patients with diarrhoea to find microsporidial spores, using the light microscopy Ryan modified trichrome stain. Microsporidia were found in one out of 56 patients, who was Cryptosporidium coinfected. Intestinal microsporidiosis seems to be less frequent in AIDS patients from Italy than in those from other countries.
Abstract: Lipophosphoglycan (LPG), a surface glycoconjugate of Leishmania promastigotes, has been reported as playing an active role in protecting the parasite within phagolysosomes, by an impairment of monocyte oxidative responses. In this study the effect of LPG on the oxidative burst of human peripheral monocytes, eosinophils and neutrophils was evaluated. Our results demonstrated that either superoxide anion (O2-) or hydrogen peroxide (H2O2) release by LPG-pretreated cells was diminished, emphasizing the ability of this glycoconjugate to impair the oxidative activity of all phagocytes.
Abstract: Protozoans of the genus Cryptosporidium may cause serious diarrhoeal illness in immunocompromised hosts and especially in HIV-infected patients. In this study we have evaluated the frequency of Cryptosporidium in stools of 51 HIV-infected patients with diarrhoea. Laboratory diagnosis of cryptosporidiosis was performed of faecal samples concentrated by a formalin-ether sedimentation technique and stained by a modified cold Ziehl-Neelsen method. Results demonstrated that 17 (33.3%) of these patients were infected with Cryptosporidium. Moreover, Cryptosporidium infection was the first clinical marker of AIDS in 7 cases. Our data show that the prevalence of this parasitosis in HIV-infected people seems to be higher in our region (Apulia, South Italy), compared to other areas of the world.
Abstract: We examined 444 dogs by visiting farms and peripheral districts on the Gargano promontory (Province of Foggia, Apulia, South Italy). Clinical examination and immunofluorescence antibody test for leishmaniasis were performed. Bone marrow and lymph node samples obtained from 25 dogs with positive serological test were cultured on Tobie-Evans medium. The results obtained show both a higher seropositivity rate for canine leishmaniasis (14.4%) and a higher percentage of asymptomatic dogs with positive serological test (53.1%) compared to previous research in the same area. Seven strains isolated from infected dogs belonged to Leishmania infantum species zymodeme Montpellier 1. None of 82 sera of humans living in close contact with infected dogs were positive for leishmaniasis.
Abstract: Bacterial lipopolysaccharides (LPS) or endotoxins are potent triggers of the cytokine (CK) cascade. These CKs are immune mediators which produce many biological effects and could play a detrimental rather than beneficial role in the host. In this review emphasis will be placed on the participation of two CKs, tumor necrosis factor [TNF-alpha and interleukin (IL-1) beta], in the pathogenetic development of HIV infection. We have found that TNF and IL-1 circulate in exaggerated amounts in the blood of HIV-infected subjects from the earliest phases of infection. Furthermore, we have observed a strict correlation between plasma LPS and IL-1 beta levels, thus indicating that endotoxins could account for the production of CKs in the course of HIV infection. Finally, the demyelinating role of TNF-alpha either in experimental models or in the course of AIDS dementia complex is outlined.
Abstract: In sera of 35 dogs with generalised leishmaniasis elevated levels of circulating immune complexes were detected by the 125I-C1q binding test. Furthermore, anti-heart muscle and anti-smooth muscle antibodies were detected by an indirect immunofluorescence technique in sera of infected dogs.
Abstract: The causes of the loosening of a hip prosthesis may be inflammatory, mechanical, immunitary or due to detritus. A bacteriological and histological study, in cases where further surgery was needed, may clearly contribute to the aetiopathogenesis of failure. Loose hip prostheses removed at operation were studied from a bacteriological and, in particular, histological point of view. There results were then correlated to clinical, radiographic and laboratory data obtained during follow-up. Inflammation proved to be a constant histological finding. The hyalinosis observed in 22 cases revealed significant analogies with histological pictures of silicotic granuloma, for which an immunitary pathogenesis is the current hypothesis. Thus, it is our belief that this correlation is suggestive of an immunitary based approach in cases where hip prostheses fail due to loosening.
Abstract: A case of cryptosporidiosis in a male homosexual man with AIDS is reported. The clinical manifestations were anorexia, mild fever, abdominal pain and profuse watery diarrhea. The diagnosis was made in fecal smears by using a modified Ziehl-Neelsen method and a safranin-methylene blue staining technique.
Abstract: Fifteen dogs with generalised leishmaniasis have been evaluated for their monocyte capacity to ingest Leishmania promastigotes. These cells displayed a lower phagocytic ability when compared to monocytes recovered from normal dogs. Moreover, sera from Leishmania-infected dogs caused a remarkable decrease of monocyte phagocytic ability in healthy dogs, whereas sera from healthy dogs could restore monocyte phagocytosis in sick animals. Similar results were seen when Candida albicans was used as stimulant. Interestingly, supernatants from polyethylenglycole (PEG) treated sera from Leishmania-infected dogs did not inhibit monocyte phagocytosis either in the autologous or in the homologous (healthy dogs) system. This suggests a possible role for serum factor(s) in the impairment of phagocytosis in Leishmania infection.
Abstract: 175 sera from children and 101 sera from adults both with respiratory illness of unknown origin were collected between September 1980 and February 1983 and studied by using the indirect immunofluorescence test for Legionnaires' disease. By using the criteria of the Center for Disease Control for a positive (fourfold increase in titer over 128 during convalescence) or presumptive (single titer greater than or equal to 256) serological test, 8 samples from adults (7.92%) and 11 samples from children (6.27%) gave a positive result. These results suggest that the frequency of Legionnaires' disease is similar in both adults and children.
Abstract: Among several children admitted in Hospital with febrile acute pneumonia, it has been found - by indirect immunofluorescence assay a significant seroconversion for Legionella pneumophila serogroup 1, compatible with a recent infection, in an 18-month-old boy. The clinical course of the disease was favourable, and a prompt recovery as observed after administration of an antibiotic (cefuroxime) stable to the bacterial beta-lactamase.
Abstract: The Authors on the basis of most recent references, expose the epidemiological, cultural, biochemical and taxonomical characteristics of microorganisms of the genus Serratia. In addition the most recent data regarding the typing and the sensitivity to antibacterial agents are discussed.
Abstract: Some culture filtrates or enterotoxin preparations from enterobacteria that activate the adenylate cyclase system (vibrio cholerae, LT fraction from escherichia coli and klebsiella pneumoniae, shigella dysenteriae type 1) exibit an inhibiting effect on ADP-induced platelet aggregation, while other enterotoxin preparations not effective on adenylate cyclase system, don't interfere with this model. The A. propose the platelet aggregation as cellular assay to detect enterotoxin fractions effective upon adenylate cyclase system.
Abstract: After some notices on the Limulus amebocyte lysate test the AA. expose the results of an investigation for in vitro pyrogen detection in parenteral preparations.
