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1.
PLoS One ; 15(9): e0238188, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32870947

RESUMO

Visceral leishmaniasis caused by the protozoan Leishmania infantum is a zoonosis. The domestic dog is the primary reservoir in urban areas. This study aimed to evaluate the frequency, active infection and load of L. infantum in the genital tract of male and female dogs seropositive for this parasite, as well as to identify histological genital alterations associated with this protozoan. We studied 45 male and 25 female L. infantum-seropositive noncastrated dogs from the same endemic area in Brazil. Tissue samples from the testis, epididymis, prostate, vulva, vagina, and uterus were examined by singleplex qPCR and parasitological tests (histopathology, immunohistochemistry, and parasitological culture). The latter were performed for the detection of active infection (parasites able to multiply and to induce lesions). Forty-four (98%) males and 25 (100%) females were positive for L. infantum in the genital tract (epididymis: 98%; vulva: 92%; vagina: 92%; testis: 91%; uterus: 84%; prostate: 66%). Active infection in the genital tract was confirmed in 69% of males and 64% of females (32% in the uterus). Parasite loads were similar in the testis, vulva, epididymis and vagina and lower in the prostate. Only the parasite load in the vagina was significantly associated with the number of clinical signs. Granulomatous inflammation predominated in all organs, except for the prostate. Only in the testis and epididymis was the inflammatory infiltrate significantly more intense among dogs with a higher parasite load in these organs. The high frequency, detection of active infection and similarity of L. infantum loads in the genital tract of infected males and females suggest the potential of venereal transmission of this parasite by both sexes and of vertical transmission by females in the area studied. Additionally, vertical transmission may be frequent since active L. infantum infection was a common observation in the uterus.


Assuntos
Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Genitália/parasitologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/veterinária , Animais , Cães , Doenças Endêmicas/veterinária , Feminino , Leishmaniose Visceral/epidemiologia , Masculino , Prevalência
2.
Mem Inst Oswaldo Cruz ; 115: e200140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32965329

RESUMO

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(ß1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.


Assuntos
Glicoesfingolipídeos , Leishmania infantum/fisiologia , Animais , América Central , Honduras , Humanos , Macrófagos/imunologia , Masculino , Camundongos
3.
Mol Immunol ; 127: 95-106, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32949849

RESUMO

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.


Assuntos
Leishmania infantum/fisiologia , Leishmania/fisiologia , Leishmaniose/prevenção & controle , Leishmaniose/parasitologia , Plasmídeos/metabolismo , Toxinas Biológicas/metabolismo , Transfecção , Animais , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Celular , Imunidade Humoral , Imunização , Leishmania/patogenicidade , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/patogenicidade , Leishmaniose/imunologia , Estágios do Ciclo de Vida , Camundongos Endogâmicos BALB C , Parasitos/metabolismo , Parasitos/patogenicidade , Proteínas de Protozoários/metabolismo , Virulência
4.
PLoS Negl Trop Dis ; 14(7): e0008396, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32722702

RESUMO

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Microtúbulos/parasitologia , Animais , Endossomos/metabolismo , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/metabolismo , Camundongos , Microtúbulos/metabolismo , Fagossomos/metabolismo , Células RAW 264.7
5.
Exp Parasitol ; 216: 107939, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32535115

RESUMO

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.


Assuntos
Glucosilceramidase/antagonistas & inibidores , Leishmania/fisiologia , Macrófagos/parasitologia , Fagocitose , Animais , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Doença de Gaucher/complicações , Doença de Gaucher/fisiopatologia , Glucosilceramidase/efeitos dos fármacos , Glucosilceramidase/genética , Inositol/análogos & derivados , Inositol/farmacologia , Leishmania infantum/fisiologia , Leishmania major/fisiologia , Leishmania mexicana/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fagocitose/efeitos dos fármacos
7.
Parasitol Res ; 119(7): 2245-2255, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32447515

RESUMO

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Microautofagia/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Doenças do Cão/parasitologia , Cães , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Leishmaniose Visceral/parasitologia , Pele/parasitologia , Ativação Transcricional , Regulação para Cima/imunologia , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Vet Parasitol ; 280: 109058, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32200198

RESUMO

The clinical manifestations most frequently observed in cats with leishmaniasis caused by Leishmania infantum are cutaneous alterations, which suggest a high parasitic load in the skin and the possibility of infecting a vector. This study evaluated the infectiousness of to phlebotomine sand flies cats infected with L. infantum. A total of 12 cats with infection by L. infantum from the city of Teresina, Piauí, Brazil, were included in the study. Cats were diagnosed by direct visualization of the parasite. Laboratory-bred insects, free from infection by Leishmania spp. were offered a blood meal for 60 min on cats infected with L. infantum. On the fifth and sixth day after the blood meal, flies were dissected to assess promastigote forms of the parasite in the digestive system. Eight cats (67 %) were able to infect the vectors. The frequency of infected insects per cat ranged 0.0-94.4%. The mean frequency of insects feeding on cats was 95.2 %. Large numbers of the parasite were observed per insect, but were not quantified. The result confirm that cats are able to infect L. longipalpis, indicating that cats are part of the epidemiological chain of VL, acting as reservoir of the disease.


Assuntos
Doenças do Gato/transmissão , Insetos Vetores/parasitologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/veterinária , Psychodidae/parasitologia , Animais , Brasil , Gatos , Feminino , Leishmaniose Visceral/transmissão , Masculino
9.
J Immunol Res ; 2020: 9602576, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32211445

RESUMO

The leishmanin skin test (LST) is an in vivo technique commonly used to evaluate the Leishmania-specific cellular immune response in dogs. However, information regarding the local immune response in LST-positive reactions is scarce. We examined the pattern of toll-like receptor 2 (TLR2), TLR4, TLR7, interleukin- (IL-) 10, interferon gamma (IFN-γ), and (program death ligand) PD-L1 gene expression in LST-positive reactions and paired normal-looking skin of nine infected Ibizan hound dogs. Healthy skin from ten seronegative dogs from a nonendemic area was analysed as a negative control. Immune gene expressions were examined by quantitative PCR (qPCR) analysis. LST-positive reactions presented significant upregulation of TLR2, TLR4, IL-10, IFN-γ, and PD-L1 and downregulation of TLR7 when compared with healthy skin of seronegative control dogs from a nonendemic area. All transcripts but TLR7 were significantly higher in LST-positive reaction than in paired normal-looking skin of Ibizan hound. The expression profile of immune genes in LST-positive reactions was similar to that previously observed in clinically lesioned skin of mildly diseased dogs with papular dermatitis due to Leishmania infantum infection. This data provide additional support for the important role of TLRs in canine leishmaniosis.


Assuntos
Antígeno B7-H1/metabolismo , Cães/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leishmania infantum/fisiologia , Leishmaniose/diagnóstico , Pele/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Animais , Anticorpos Antiprotozoários/metabolismo , Antígenos de Protozoários/metabolismo , Antígeno B7-H1/genética , Interferon gama/genética , Interleucina-10/genética , Testes Cutâneos , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 7 Toll-Like/genética , Regulação para Cima
10.
Res Vet Sci ; 130: 19-25, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32109759

RESUMO

Immune control of Leishmania infantum, the causative agent of most canine leishmaniosis (CanL), requires a balancing act between inflammatory and regulatory responses. This balance is specifically between the proinflammatory T helper 1 type (Th1) CD4+ T cells that are responsible for controlling parasite replication and T regulatory 1 cells which mediate an immunosuppressive, regulatory, response needed to dampen overabundant inflammation but if predominant, result in CanL progression. How this delicate immune cell interaction occurs in the dog will be highlighted in this review, focusing on the progressive changes observed within myeloid lineage cells (predominantly macrophages), B cells and T cells. After exposure to parasites, macrophages should become activated, eliminating L. infantum through release of reactive oxygen species. Unfortunately, multiple parasite and host factors can prevent macrophage activation allowing parasites to persist within them. T cells balance between a productive TH1 type CD4+ response capable of producing IFN-γ which aids macrophage activation versus T cell exhaustion which reduces T cell proliferation, IFN-γ production and allows parasite expansion within macrophages. Neutrophils and Th17 cells add to the inflammatory state, aiding in parasite removal, but also leading to pathology. A regulatory B cell population increases IL-10 production and down regulates the TH1 response allowing parasite growth. All of these immune challenges affect the balance between progression to clinical disease and maintaining sub-clinical disease. Vaccines and immunotherapies targeted at recovering or maintaining T and B cell function can be important factors in mending the immune balance required to survive CanL.


Assuntos
Linfócitos B/imunologia , Doenças do Cão/imunologia , Leishmaniose Visceral/veterinária , Macrófagos/imunologia , Linfócitos T/imunologia , Animais , Cães , Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia
11.
PLoS Negl Trop Dis ; 14(1): e0008021, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961868

RESUMO

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/imunologia , Interleucina-12/administração & dosagem , Interleucina-15/administração & dosagem , Leishmaniose Visceral/imunologia , Animais , Doenças do Cão/genética , Doenças do Cão/parasitologia , Cães , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia
12.
PLoS Negl Trop Dis ; 14(1): e0007949, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961876

RESUMO

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.


Assuntos
Inflamassomos/imunologia , Leishmania infantum/fisiologia , Leishmaniose/genética , Monócitos/imunologia , Monócitos/parasitologia , Caspase 1/genética , Caspase 1/imunologia , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/imunologia , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Células THP-1 , Transcriptoma , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
14.
Transbound Emerg Dis ; 67(3): 1113-1118, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31765072

RESUMO

Vertical transmission of Leishmania infantum was demonstrated in domestic mice captured close to the home of a patient with leishmaniasis. Leishmania infantum DNA was detected in 88.9% of synanthropic Mus musculus adult rodents and 29.2% of their unborn foetuses. Mother-to-infant transmission was observed in all females whose gestational stage was sufficiently advanced to allow foetal analysis (foetal length 2-2.5 cm). The infection rate in foetal samples ranged from 11.1% to 50.0%, with parasite loads of up to 6,481 parasites/5 mg tissue. A low density of Phlebotomus perniciosus was also found (0.2 specimen/CDC trap). Six infected mice captured in March were only 1.5 months old and could thus not have had contact with the vector. Vertical transmission thus appears to play a greater role in the spread of leishmaniasis than previously thought, particularly since rodents are natural hosts for the parasite and are prolific in nature.


Assuntos
Transmissão Vertical de Doença Infecciosa/veterinária , Leishmania infantum/fisiologia , Leishmaniose/veterinária , Phlebotomus/parasitologia , Animais , Feminino , Leishmania infantum/genética , Leishmaniose/transmissão , Masculino , Camundongos , Roedores
15.
Med Vet Entomol ; 34(2): 240-243, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31769060

RESUMO

Dogs are the reservoir host of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). Both subclinically-infected and sick animals can be infectious to competent phlebotomine vectors. The degree and duration of insecticidal efficacy of an oral dose of fluralaner (Bravecto®; Merck Animal Health) was determined in dogs exposed to bites of Phlebotomus perniciosus (Diptera: Psychodidae), a main Mediterranean vector of VL. Twelve dogs allocated to two groups of six animals each were included in a parallel-group designed, negative-controlled, randomized, blinded, single-centre efficacy study. Group 2 was treated with fluralaner on day 0, and sand-fly exposure of both groups was performed on days 1, 28 and 84. Viability of blood-fed females was assessed up to 96 h after exposure and efficacy was measured as the survival rate of specimens fed on Group 2 versus those fed on Group 1. A mortality of 100% was recorded at 24 h in females fed on Group 2 at both days 1 and 28. Significant insecticidal efficacy was still observed on day 84, with > 50% mortality recorded by 48 h post blood meal in Group 2. Fluralaner treatment of dogs represents a promising and affordable method for reducing the pool of infected vectors in endemic settings of zoonotic VL.


Assuntos
Doenças do Cão/prevenção & controle , Controle de Insetos , Insetos Vetores , Inseticidas , Isoxazóis , Leishmaniose Visceral/veterinária , Phlebotomus , Administração Oral , Animais , Doenças do Cão/parasitologia , Cães , Feminino , Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Masculino , Fatores de Tempo
16.
Vet Parasitol ; 277: 109015, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31874403

RESUMO

Canine leishmaniosis (CanL)-associated chronic kidney disease is a leading cause of morbidity and mortality in Mediterranean countries. Novel renal biomarkers, such as serum symmetric dimethylarginine (sSDMA), may be useful surrogates for the detection of renal functional impairment. The objectives of this study were to investigate sSDMA concentrations in dogs with CanL, with and without azotemia, and to establish any potential association with the prevalence and severity of proteinuria, with the prevalence of decreased urine specific gravity and with the LeishVet clinical stages of CanL. Serum samples from 68 dogs with CanL (50 nonazotemic and 18 azotemic) and 17 healthy dogs were retrospectively examined. Increased sSDMA was documented in 26 % of dogs with CanL without azotemia and in 83.3 % of dogs with azotemia. Serum SDMA was significantly higher in azotemic compared to nonazotemic dogs and was associated with the presence and severity of proteinuria, the decreased urine specific gravity and the advanced clinical stages of CanL. The results of the present study indicate that sSDMA may be a useful adjunct to serum creatinine and urine protein/creatinine ratio for the detection of CanL-associated nephropathy, but it is of limited value for distinguishing among the LeishVet clinical stages of CanL.


Assuntos
Arginina/análogos & derivados , Doenças do Cão/diagnóstico , Leishmaniose Visceral/complicações , Leishmaniose Visceral/diagnóstico , Insuficiência Renal Crônica/etiologia , Animais , Arginina/sangue , Azotemia/veterinária , Biomarcadores/sangue , Doenças do Cão/sangue , Cães , Leishmania infantum/fisiologia , Insuficiência Renal Crônica/sangue , Estudos Retrospectivos
17.
Front Immunol ; 10: 2749, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849951

RESUMO

Visceral leishmaniasis (VL) caused by Leishmania donovani and L. infantum is a potentially fatal disease. To date there are no registered vaccines for disease prevention despite the fact that several vaccines are in preclinical development. Thus, new strategies are needed to improve vaccine efficacy based on a better understanding of the mechanisms mediating protective immunity and mechanisms of host immune responses subversion by immunopathogenic components of Leishmania. We found that mice vaccinated with CPA162-189-loaded p8-PLGA nanoparticles, an experimental nanovaccine, induced the differentiation of antigen-specific CD8+ T cells in spleen compared to control mice, characterized by increased dynamics of proliferation and high amounts of IFN-γ production after ex vivo re-stimulation with CPA162-189 antigen. Vaccination with CPA162-189-loaded p8-PLGA nanoparticles resulted in about 80% lower parasite load in spleen and liver at 4 weeks after challenge with L. infantum promastigotes as compared to control mice. However, 16 weeks after infection the parasite load in spleen was comparable in both mouse groups. Decreased protection levels in vaccinated mice were followed by up-regulation of the anti-inflammatory IL-10 production although at lower levels in comparison to control mice. Microarray analysis in spleen tissue at 4 weeks post challenge revealed different immune-related profiles among the two groups. Specifically, vaccinated mice were characterized by similar profile to naïve mice. On the other hand, the transcriptome of the non-vaccinated mice was dominated by increased expression of genes related to interferon type I, granulocyte chemotaxis, and immune cells suppression. This profile was significantly enriched at 16 weeks post challenge, a time-point which is relative to disease establishment, and was common for both groups, further suggesting that type I signaling and granulocyte influx has a significant role in disease establishment, pathogenesis and eventually in decreased vaccine efficacy for stimulating long-term protection. Overall, we put a spotlight on host immune networks during active VL as potential targets to improve and design more effective vaccines against disease.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Cisteína Proteases/imunologia , Leishmania donovani/fisiologia , Leishmania infantum/fisiologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Fígado/imunologia , Nanopartículas/administração & dosagem , Peptídeos/imunologia , Proteínas de Protozoários/imunologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Cisteína Proteases/química , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Fígado/parasitologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Nanopartículas/química , Carga Parasitária , Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas de Protozoários/química
18.
PLoS One ; 14(12): e0226192, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31825987

RESUMO

Visceral Leishmaniasis is a chronic zoonosis and, if left untreated, can be fatal. Infected dogs have decreased cellular immunity (Th1) and develop a potent humoral response (Th2), which is not effective for elimination of the protozoan. Immune response can be modulated by microRNAs (miRNAs), however, characterization of miRNAs and their possible regulatory role in the spleen of infected dogs have not been done. We evaluated miRNA expression in splenic leukocytes (SL) from dogs naturally infected with Leishmania infantum and developing leishmaniasis (CanL; n = 8) compared to healthy dogs (n = 4). Microarray analysis showed increased expression of miR 21, miR 148a, miR 7 and miR 615, and downregulation of miR 150, miR 125a and miR 125b. Real-time PCR validated the differential expression of miR 21, miR 148a and miR 615. Further, decrease of miR 21 in SL, by means of transfection with a miR 21 inhibitor, increased the IL-12 cytokine and the T-bet/GATA-3 ratio, and decreased parasite load on SL of dogs with CanL. Taken together, these findings suggest that L. infantum infection alters splenic expression of miRNAs and that miR 21 interferes in the cellular immune response of L. infantum-infected dogs, placing this miRNA as a possible therapeutic target in CanL.


Assuntos
Doenças do Cão/diagnóstico , Interleucina-12/metabolismo , Leishmaniose Visceral/diagnóstico , Leucócitos/metabolismo , MicroRNAs/metabolismo , Baço/metabolismo , Animais , Antagomirs/metabolismo , Anticorpos Monoclonais/imunologia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Regulação para Baixo , Fator de Transcrição GATA3/metabolismo , Imunidade Celular , Interleucina-12/antagonistas & inibidores , Leishmania infantum/imunologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leucócitos/citologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Baço/imunologia , Proteínas com Domínio T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima
19.
PLoS Negl Trop Dis ; 13(11): e0007832, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31751334

RESUMO

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field.


Assuntos
Doenças do Cão/diagnóstico , Imunoensaio/métodos , Insetos Vetores/fisiologia , Leishmaniose/veterinária , Phlebotomus/fisiologia , Animais , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Cães , Feminino , Insetos Vetores/parasitologia , Leishmania infantum/fisiologia , Leishmaniose/sangue , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Camundongos Endogâmicos BALB C , Phlebotomus/parasitologia
20.
J Vector Borne Dis ; 56(2): 127-133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31397388

RESUMO

Background & objectives: Cutaneous leishmaniasis (CL) is widespread in the tropical and subtropical regions of the world including, Tukey. Environmental determinants for the CL endemic areas in Turkey are relatively poorly understood. The aim of the present study was to develop a model based on ecological niche modeling (ENM) to predict the distribution of CL in endemic areas of Adana Province in Turkey. Methods: The environmental data from different sources were extracted and information on 1831 native CL cases, obtained from the Provincial Health Directorate of Adana were recorded. The location information obtained from the Ministry of Health database were used for modeling the current probability of CL occurrence and predicting its future distribution using ENM analyses. ArcGIS and MaxEnt models were used to explore the ecological conditions of the disease. Results: According to the MaxEnt model, the area under the curve (AUC) values for the current and projected future of CL were 0.868 and 0.867, respectively. The environmental variables, Bio1 (Annual mean temperature), Bio4 (Temperature seasonality) and DEM (Digital elevation model) were found to be associated with the presence of human cases of Leishmania infantum for both the time periods in the study area. Interpretation & conclusion: The AUC curves and risk map generated by the ENM model indicate that the future status of CL is likely to be stable in the northern part of Adana, but the southern part will be affected by climate changes (change of temperature) with a large number of patient-reporting. The results of the study could be used as a reference for CL and vector control studies. The ENM could be useful for researchers in vector control studies and better understanding of the epidemiology of vector-borne diseases in a specific area.


Assuntos
Ecossistema , Leishmania infantum/fisiologia , Leishmaniose Cutânea/epidemiologia , Modelos Biológicos , Animais , Área Sob a Curva , Humanos , Insetos Vetores/parasitologia , Phlebotomus/parasitologia , Temperatura , Turquia/epidemiologia
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