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1.
Artigo em Inglês | MEDLINE | ID: mdl-33533815

RESUMO

Visceral leishmaniasis (VL) is a neglected tropical disease caused by the Leishmania infantum parasite. The protozoan is able to infect several domestic and wild mammals. Since the first report on Leishmania spp. infection in horses in South America, leishmaniasis in equids has been highlighted in Brazil. A molecular epidemiological survey was carried out to verify the occurrence of Leishmania spp. DNA in horses and donkeys, in leishmaniases endemic areas in Sao Paulo State, Brazil. To this end, blood samples were obtained from 107 horses and 36 donkeys and subjected to DNA extraction followed by PCR targeting the ITS-1 region. Among the horses and donkeys, 1.87% (2/107) and 8.33% (3/36) were positive by PCR, respectively. The DNA sequencing of the ITS-1 amplification products confirmed L. infantum DNA in these animals. Our results suggest that horses and donkeys from non-VL and VL endemic areas of São Paulo State may be infected by the parasite.


Assuntos
Equidae/sangue , Cavalos/sangue , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Animais , Brasil , DNA , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase
2.
Am J Trop Med Hyg ; 103(5): 1930-1933, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32959759

RESUMO

There has been increased interest in using metagenomic next-generation sequencing as an unbiased approach for diagnosing infectious diseases. We describe a 61-year-old man on fingolimod therapy for multiple sclerosis with an extensive travel history who presented with 7 months of fevers, night sweats, and weight loss. Peripheral blood tests showed pancytopenia and abnormal acute phase reactants. A bone marrow aspirate showed the presence of numerous intracellular and extracellular amastigotes consistent with visceral leishmaniasis (VL). Metagenomic sequencing of the bone marrow aspirate confirmed Leishmania infantum, a species widely reported in the Mediterranean region. This correlated with acquisition of VL infection during the patient's most recent epidemiological exposure in southern Italy 12 months prior. This case demonstrates the potential application of metagenomic sequencing for identification and speciation of Leishmania in cases of VL; however, further assessment is required using other more readily obtained clinical samples such as blood.


Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Metagenômica , Medula Óssea/parasitologia , Humanos , Hospedeiro Imunocomprometido , Itália , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Masculino , Pessoa de Meia-Idade , Viagem
3.
Parasitol Res ; 119(10): 3541-3548, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32803333

RESUMO

The aim of this study was to evaluate, through qPCR, the prevalence of parasitemia in sick kennel dogs naturally infected by canine leishmaniasis. An evaluation of daily changes of the parasitic load in peripheral blood was also performed. A comprehensive clinical examination and the collection of several samples (blood, lymph node, skin, and conjunctiva) were performed in 140 dogs living in an endemic area. Among these, only the dogs with clinically evident leishmaniasis were enrolled (39/140; 27.9%). Twelve (30.8%) out of 39 showed parasitemia, with a low load (median: 4 Leishmania/ml) despite a high lymph node parasite load (median: 4000 Leishmania/ml) and high IFAT titers (≥ 1:640). Seven sick dogs were sampled every 4 h for 6 times during a 24-h period, in order to obtain light- and dark-span samples. Only one (14.3%) out of the seven serial sampled dogs showed Leishmania DNA in the peripheral blood in two samples (2/42; 4.8%). Surprisingly, Leishmania DNA was also detected in the peripheral blood of asymptomatic dogs, negative to both serology and PCR performed on samples other than blood (6/101; 5.9%). The present study confirms that in canine leishmaniasis parasitemia is uncommon and even transitory. Even if recommended, microscopic examination is confirmed as a low sensitivity method with a lower diagnostic utility in canine leishmaniasis than qPCR. Moreover, circulating Leishmania DNA can be found even in healthy dogs. This finding is important in clinical practice because in endemic areas it suggests a transfusion risk and a possible transmission to the vector.


Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Parasitemia/veterinária , Animais , DNA de Protozoário/sangue , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Carga Parasitária/veterinária , Parasitemia/epidemiologia , Parasitemia/parasitologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária
4.
PLoS Negl Trop Dis ; 14(7): e0007986, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716941

RESUMO

Genetic polymorphisms in natural Leishmania populations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition of Leishmania infantum in naturally infected dogs by genotyping parasites infecting different tissues with or without in vitro expansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48 L. infantum cultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A-I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q ≥ 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused by L. infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the effects of multiple infection on the host immune response and disease dynamics and treatment.


Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Cães , Feminino , Variação Genética , Genótipo , Leishmania infantum/classificação , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Masculino , Repetições de Microssatélites , Filogenia
5.
Exp Parasitol ; 216: 107941, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622940

RESUMO

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Medula Óssea/parasitologia , Biologia Computacional , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Imunoglobulina G/sangue , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Protozoários/química , Sensibilidade e Especificidade , Alinhamento de Sequência , Testes Sorológicos , Baço/parasitologia , Adulto Jovem
6.
PLoS Negl Trop Dis ; 14(7): e0008465, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32706789

RESUMO

BACKGROUND: Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory. CONCLUSIONS/SIGNIFICANCE: Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiology, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts.


Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Animais , Doenças do Cão/epidemiologia , Cães , Humanos , Itália/epidemiologia , Leishmania infantum/classificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia
7.
Rev Bras Parasitol Vet ; 29(2): e003520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520088

RESUMO

Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.


Assuntos
Doenças do Gato/parasitologia , Leishmania braziliensis/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Gatos , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Filogenia , Reação em Cadeia da Polimerase/veterinária
8.
Acta Trop ; 207: 105492, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32298655

RESUMO

Phlebotomine sand flies are vectors of Leishmania parasites causing different clinical forms of leishmaniases that represent a serious emerging public health problem worldwide. Mexico harbours a large diversity of sand flies, yet only six species have been considered suspected vectors of Leishmania. The disease has been recorded in 25 states, where the State of Veracruz has recorded the highest number of cases with leishmaniases, although no systematic or epidemiological surveillance studies of Leishmania vectors have been carried out in the region. For that reason, the aim of this study was to perform the molecular detection of Leishmania DNA in phlebotomine sand flies collected from a humid tropical region in Veracruz. We confirmed the presence of Leishmania DNA in eight sand fly species. Sand flies with the highest infection were Psathyromyia aclydifera and Pychodopygus panamensis. This is also the first report of Leishmania DNA in Psathyromyia aclydifera, Psathyromyia carpenteri, Dampfomyia beltrani and Brumptomyia mesai. Our findings highlight the importance of entomological surveys and epidemiological studies, since they enable to determine whether sand fly species may be potential Leishmania vectors in a given area of the Mexico, as transmission dynamics can vary in the different regions.


Assuntos
DNA de Protozoário/análise , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Masculino
9.
Acta Trop ; 207: 105495, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32305295

RESUMO

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.


Assuntos
Doenças do Cão/diagnóstico , Cães/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase/métodos , Animais , DNA de Cinetoplasto/genética , Feminino , Leishmania infantum/genética , Masculino
10.
Parasitol Res ; 119(5): 1683-1690, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32285265

RESUMO

The diagnosis of canine visceral leishmaniasis (CVL) has been a problem for public health services due to the variety of clinical signs similar to other diseases and low sensitivity and specificity of available tests. In this sense, our main objective was to develop a simple, rapid, and accurate quantitative real-time PCR (qPCR) diagnosis for CVL. Thus, low-invasive samples from bone marrow (BM), popliteal lymph nodes (PLN), and conjunctival swabs (CS) were selected from negative and VL-positive dogs, using as gold standard, immunological and parasitological tests performed with different tissues. Oligonucleotides for Leishmania infantum kDNA were designed and the limit of quantification and amplification efficiency of the qPCR were determined using tissue-specific standards produced with DNA from those different tissues, mixed with DNA from a known amount of L. infantum promastigotes. Endogenous control was used to validate a comparative Ct method, and tissue parasite concentrations were estimated by comparison with tissue-specific reference standard samples. The overall analysis of the qPCR data suggests the following ranking for tissue choice: PLN > BM > CS. Finally, we have concluded that this molecular approach simplifies and accelerates the quantitative diagnostic process because it is easy to perform, requiring no DNA dosing or standard curve application, and it shows good diagnostic parameters, especially when using popliteal lymph node samples.


Assuntos
Doenças do Cão/diagnóstico , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Medula Óssea/parasitologia , DNA de Cinetoplasto/genética , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/parasitologia , Linfonodos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Baço/parasitologia
11.
PLoS Negl Trop Dis ; 14(3): e0008077, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214313

RESUMO

BACKGROUND: Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs. METHODOLOGY AND FINDINGS: A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively. CONCLUSION: Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.


Assuntos
DNA/análise , Comportamento Alimentar , Conteúdo Gastrointestinal/química , Conteúdo Gastrointestinal/parasitologia , Especificidade de Hospedeiro , Leishmania infantum/isolamento & purificação , Phlebotomus/fisiologia , Animais , DNA/genética , Impressões Digitais de DNA , DNA Espaçador Ribossômico/genética , Transmissão de Doença Infecciosa , Feminino , Humanos , Leishmania infantum/genética , Masculino , Phlebotomus/parasitologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Tunísia
12.
PLoS Negl Trop Dis ; 14(3): e0008014, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126078

RESUMO

BACKGROUND: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. CONCLUSIONS: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.


Assuntos
Expressão Gênica , Marcadores Genéticos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Trato Gastrointestinal/parasitologia , Perfilação da Expressão Gênica , Análise de Sequência de RNA
13.
Rev Bras Parasitol Vet ; 29(1): e016319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049143

RESUMO

Leishmania infantum is a trypanosomatid that causes parasitic dermatopathy in dogs. Trypanosoma caninum is another trypanosomatid, which infects the skin of dogs, although cutaneous abnormalities are absent. This study aimed to investigate the occurrence of T. caninum infection and its associated cutaneous and histological changes and compare it with the occurrence of L. infantum infection in dogs. The study included 150 dogs, of which T. caninum infection was identified in 3 (2%) and L. infantum infection in 15 (10%) of them, with no association (p>0.05) of these infections with the breed, gender, age, or cutaneous abnormalities. The cutaneous abnormalities were based on 1 (4.8%) and 12 (57.1%) dogs infected by T. caninum and L. infantum, respectively. The dermatohistopathological abnormalities in the dogs infected with T. caninum included mild perivascular lymphohistioplasmacytic infiltrates in the clinically asymptomatic ones, while in those with dermatological abnormalities, acanthosis, epidermal orthokeratotic hyperkeratosis, melanomacrophages, and co-infection with Microsporum sp. and Trichophyton sp. were observed. InL. infantum infected, the histopathological findings included chronic granulomatous inflammatory infiltrates and structures compatible with amastigotes. Despite the low frequency of T. caninum infection, our findings suggest that this trypanosomatid, unlike L. infantum, does not cause any macroscopic skin abnormalities.


Assuntos
Doenças do Cão/patologia , Leishmania infantum/genética , Leishmaniose Visceral/veterinária , Trypanosoma/genética , Tripanossomíase/veterinária , Animais , Brasil/epidemiologia , Coinfecção , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Cães , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/patologia , Reação em Cadeia da Polimerase , Prevalência , Tripanossomíase/epidemiologia , Tripanossomíase/patologia
14.
Parasitol Res ; 119(4): 1381-1386, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32107620

RESUMO

The prevalence data of Leishmania infantum infection in cats are characterized by a large variability mainly attributed to the differences in diagnostic techniques. In the absence of consensus about the method of choice for diagnosing feline leishmaniosis, the performance of a new immunofluorescence antibody test (IFAT) was herein analytically described by the comparison with IFAT commonly used for the diagnosis of canine leishmaniosis (i.e., IFAT-OIE) and a laboratory enzyme-linked immunosorbent assay (ELISA). Sera of cats living in visceral leishmaniosis-endemic (n = 105) and visceral leishmaniosis-non-endemic (n = 50) areas were tested by the above methodologies and real-time PCR (qPCR). The most frequent result was represented by triple negativity to the three tests (IFAT-OIE, ELISA, and qPCR) in 42.9% and 80% cats from endemic and non-endemic areas, respectively. Bayes latent class analysis gave an output probability of 34.1% (posterior standard deviation, psd = 5.4%) of true L. infantum cases (TCL) which represent the true estimated prevalence of infection. The sensitivity of each variable contributing to define the TCL was 24% (psd = 6.3%) for qPCR, 78.8% (psd = 8.7%) for ELISA and 91.8% (psd = 5.2%) for IFAT-OIE. The probability to be a TCL was 94.5% for the sample from an endemic area. The cross-validation of the new IFAT by a logistic model correctly identified as positive 80.7% of subjects defined as TCL and negative 89.9% as not TCL, respectively, by the Bayesian model. The study results estimate a good accuracy of the IFAT in predicting cats exposed to L. infantum. Therefore, this procedure may be beneficial for screening cat populations for a better understanding of the epidemiology of feline leishmaniosis.


Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Técnica Direta de Fluorescência para Anticorpo/métodos , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Animais , Teorema de Bayes , Doenças do Gato/epidemiologia , Doenças do Gato/parasitologia , Gatos , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/genética , Leishmaniose Visceral/diagnóstico , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária
15.
Comp Immunol Microbiol Infect Dis ; 69: 101412, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31981798

RESUMO

In Côte d'Ivoire, limited information are available on vector-borne pathogens, their prevalence and distribution. Here, we assess the occurrence and diversity of canine vector-borne diseases (CVBDs) in Abidjan and Yamoussoukro cities. Blood from a total of 123 dogs were tested for Leishmania infantum and Ehrlichia canis antibodies and screened for Leishmania and Trypanosoma spp., Piroplasmida, Filariidae and Anaplasmataceae by PCR and sequencing. Among dogs, 39 % were positive for at least one pathogen. Seroprevalences were: 15.4 % and 12.2 % for L. infantum and E. canis, respectively. DNA of L. infantum and T. congolense (4.1 %), Baabesia vogeli (1.6 %), Filariidae (Dirofilaria immitis, D. repens and Acanthocheilonema reconditum) (10.6 %) has been detected. Anaplasmataceae were detected in (17.1 %) and E. canis was the only identified specie. Co-infections were observed in 13.8 % of dogs: E. canis-L. infantum co-infection was the most prevalent (4.9 %). Age, breed and sex of dogs do not seem to influence infections. Village dogs were more susceptible to CVBDs than kennel dogs (PV = 0.0000008). This study reports for the first time the presence of L. infantum, B. vogeli, A. reconditum, D. immitis and D. repens in dogs from Côte d'Ivoire and determines the prevalence and diversity of CVBD pathogens. The results indicate that human and animal pathogens are abundant in Ivoirian dogs which requires attention of veterinarians, physicians and authorities against these diseases, especially against major zoonosis such as visceral leishmaniasis (L. infantum).


Assuntos
Doenças do Cão/epidemiologia , Doenças do Cão/etiologia , Doenças Transmitidas por Vetores/epidemiologia , Doenças Transmitidas por Vetores/etiologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Costa do Marfim/epidemiologia , Dirofilaria immitis/genética , Dirofilaria immitis/imunologia , Vetores de Doenças , Doenças do Cão/sangue , Doenças do Cão/transmissão , Cães , Humanos , Leishmania infantum/genética , Leishmania infantum/imunologia , Filogenia , Reação em Cadeia da Polimerase , Vigilância em Saúde Pública , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Doenças Transmitidas por Vetores/sangue , Doenças Transmitidas por Vetores/transmissão
16.
Acta Trop ; 201: 105217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605692

RESUMO

Glycosomes of trypanosomatids are peroxisome-like organelles comprising unique metabolic features, among which the lack of the hallmark peroxisomal enzyme catalase. The absence of this highly efficient peroxidase from glycosomes is presumably compensated by other antioxidants, peroxidases of the peroxiredoxin (PRX) family being the most promising candidates for this function. Here, we follow on this premise and investigate the product of a Leishmania infantum gene coding for a putative glycosomal PRX (LigPRX). First, we demonstrate that LigPRX localizes to glycosomes, resorting to indirect immunofluorescence analysis. Second, we prove that purified recombinant LigPRX is an active peroxidase in vitro. Third, we generate viable LigPRX-depleted L. infantum promastigotes by classical homologous recombination. Surprisingly, phenotypic analysis of these knockout parasites revealed that promastigote survival, replication, and protection from oxidative and nitrosative insults can proceed normally in the absence of LigPRX. Noticeably, we also witness that LigPRX-depleted parasites can infect and thrive in mice to the same extent as wild type parasites. Overall, by disclosing the dispensable character of the glycosomal peroxiredoxin in L. infantum, this work excludes this enzyme from being a key component of the glycosomal hydroperoxide metabolism and contemplates alternative players for this function.


Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Microcorpos/metabolismo , Oxirredutases/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Camundongos , Microcorpos/genética , Oxirredutases/genética , Peroxirredoxinas/genética
17.
Acta Trop ; 201: 105178, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31606374

RESUMO

Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.


Assuntos
Leishmania infantum/genética , Leishmania mexicana/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Brasil , Cães , Humanos
18.
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
19.
Am J Trop Med Hyg ; 102(2): 289-293, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31802736

RESUMO

American visceral leishmaniasis (VL) is a vector-borne disease caused by Leishmania infantum (syn. chagasi) and transmitted by Lutzomyia longipalpis and Lutzomyia evansi phlebotomine sand flies. Dogs not only are the main host reservoirs of the parasite but also suffer the disease; therefore, canine VL (CVL) has assumed an important role in public health. In Colombia, human and CVL are restricted to two transmission foci: one in the north region (Caribbean coast) and other in the central south region (middle Magdalena River Valley). We present a CVL case involving a 2-year-old male dog with a history of lack of appetite, general weakness, and progressive loss of weight. A diagnosis of CVL was obtained using the direct parasitological examination in spleen and bone marrow samples stained with Giemsa and RT-qPCR. The infecting Leishmania species was identified as L. infantum by PCR-restriction fragment length polymorphism amplifying the Hsp70 gene from bone marrow and spleen samples and confirming by sequencing. The patient responded favorably to treatment with intramuscular meglumine antimoniate at a dose of 100 mg/kg daily for 8 weeks and oral allopurinol at a dose of 10 mg/kg every 12 hours until new indication. This is the first report of urban CVL in the city of Cali, Colombia, highlighting the need for surveillance and control programs in the municipalities of the department of Valle del Cauca, a region where VL has not been informed before. The findings also indicate the need to reinforce the surveillance programs in other rural and urban regions of the country where favorable eco-epidemiological conditions exist.


Assuntos
Doenças do Cão/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Alopurinol/uso terapêutico , Animais , Antimetabólitos/uso terapêutico , Antiprotozoários/uso terapêutico , Sequência de Bases , Cidades/epidemiologia , Colômbia/epidemiologia , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Doenças do Cão/epidemiologia , Cães , Leishmania infantum/genética , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/epidemiologia , Masculino , Antimoniato de Meglumina/uso terapêutico
20.
Rev. bras. parasitol. vet ; 29(1): e009819, 2020. graf
Artigo em Inglês | LILACS | ID: biblio-1058019

RESUMO

Abstract The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.


Resumo O objetivo deste estudo foi confirmar a emergência da leishmaniose visceral canina em Foz do Iguaçu próximo à fronteira com a Argentina e ao Paraguai, por meio do isolamento e identificação molecular de Leishmania infantum. Em um primeiro estágio de coleta de animais (2012), três amostras de aspirados de linfonodos e 46 camadas leucocitárias foram obtidas de cães soropositivos para leishmaniose. Em um segundo estágio de coleta (2013), foram coletadas amostras de camada leucocitária de 376 cães de 20 localidades próximas à fronteira com o Paraguai, rio Paraná, centro e periferia da cidade. Seis isolados foram obtidos, quatro da primeira etapa (4/49) e dois da segunda etapa (2/376); estes isolados foram submetidos à amplificação com iniciadores K26F/R, e a análise de sua sequência confirmou a espécie como L. infantum. A autoctonia dos casos foi confirmada, pois 100% dos cães nunca haviam saído da cidade. O estudo confirma a emergência de leishmaniose visceral canina no Paraná com identificação de L. infantum em cães da cidade de Foz do Iguaçu. Assim, medidas de controle devem ser elaboradas e implementadas por órgãos públicos e instituições de pesquisa do Brasil, Argentina e Paraguai em parceria com o objetivo de controlar a disseminação de zoonoses e os casos humanos de LV.


Assuntos
Animais , Cães , DNA de Protozoário/genética , Leishmania infantum/genética , Doenças do Cão/parasitologia , Leishmaniose Visceral/veterinária , Brasil/epidemiologia , Leishmania infantum/isolamento & purificação , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/epidemiologia
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