Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis

BackgroundThe sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs.Methodology/Principal findingsWe carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions.ConclusionsThe results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.     

Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis

Background

Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania.

Results

Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods.

Conclusions

These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

     

Potential distributions of phlebotomine sandfly vectors of human visceral leishmaniasis caused by Leishmania infantum in Morocco

Zoonotic visceral leishmaniasis is a common vector-borne systemic disease caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). In Morocco the situation is complex: many sandfly species have been collected in areas in which the disease is endemic, but only Phlebotomus ariasi, Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) have been confirmed to have vectorial roles. The objective of the present study was to ascertain the potential distribution of L. infantum and its vectors in Morocco, using ecological niche modelling. Vector records were obtained from field collections of the Laboratory team and from previously published entomological observations. Epidemiological data for L. infantum modelling were obtained from Moroccan Ministry of Health reports. The jackknife test indicated that the bioclimatic variables with the greatest influence on model development for all species were annual precipitation and precipitation in the driest quarter of the year. MaxEnt model representations for sandfly species that act as vectors of L. infantum showed the widespread geographic distribution of these species in Morocco, specifically in northern and central Morocco, where foci of visceral leishmaniasis are found. The ecological niche modelling points out areas in which the probability of occurrence of these species is higher. This information should be considered as a starting point for further research to fully elucidate the ecology and epidemiology of these species, as well as of the pathogens they transmit.     

Potential of Artesunate in the treatment of visceral leishmaniasis in dogs naturally infected by Leishmania infantum: Efficacy evidence from a randomized field trial

Leishmaniasis is among the world’s most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.     

Comparative analysis of the gut microbiota of sand fly vectors of zoonotic visceral leishmaniasis (ZVL) in Iran; host-environment interplay shapes diversity

The development of Leishmania parasites within sand fly vectors occurs entirely in the insect gut lumen, in the presence of symbiotic and commensal bacteria. The impacts of host species and environment on the gut microbiome are currently poorly understood. We employed MiSeq sequencing of the V3-16S rRNA gene amplicons to characterize and compare the gut microbiota of field-collected populations of Phlebotomus kandelakii, P. perfiliewi, P. alexandri, and P. major, the primary or secondary vectors of zoonotic visceral leishmaniasis (ZVL) in three distinct regions of Iran where ZVL is endemic. In total, 160,550 quality-filtered reads of the V3 region yielded a total of 72 operational taxonomic units (OTUs), belonging to 23 phyla, 47 classes, 91 orders, 131 families, and 335 genera. More than 50% of the bacteria identified were Proteobacteria, followed by Firmicutes (22%), Deinococcus-Thermus (9%), Actinobacteria (6%), and Bacteroidetes (5%). The core microbiome was dominated by eight genera: Acinetobacter, Streptococcus, Enterococcus, Staphylococcus, Bacillus, Propionibacterium, Kocuria, and Corynebacterium. Wolbachia were found in P. alexandri and P. perfiliewi, while Asaia sp. was reported in P. perfiliewi. Substantial variations in the gut bacterial composition were found between geographically distinct populations of the same sand fly species, as well as between different species at the same location, suggesting that sand fly gut microbiota is shaped by both the host species and geographical location. Phlebotomus kandelakii and P. perfiliewi in the northwest, and P. alexandri in the south, the major ZVL vectors, harbor the highest bacterial diversity, suggesting a possible relationship between microbiome diversity and the capacity for parasite transmission. In addition, large numbers of gram-positive human or animal pathogens were found, suggesting that sand fly vectors of ZVL could pose a potential additional threat to livestock and humans in the region studied. The presence of Bacillus subtilis, Enterobacter cloacae, and Asaia sp suggests that these bacteria could be promising candidates for a paratransgenesis approach to the fight against Leishmaniasis.     

Apoptosis of Leishmania (Leishmania) chagasi amastigotes in hamsters infected with visceral leishmaniasis

Apoptosis in amastigotes from hamsters infected with visceral leishmaniasis was absent 30-day post-infection but appeared 90-day post-infection in the liver and spleen, as analysed using the TUNEL method. Necrosis was not present in these tissues and the nuclei of macrophages harbouring apoptotic amastigotes were preserved. Amastigote DNA fragmentation was demonstrated using agarose gel electrophoresis. DNA fragmentation was evident 90-day post-infection, coinciding with the occurrence of apoptosis of amastigotes in the tissues. Apoptosis of Leishmania amastigotes in vivo may constitute a mechanism that regulates growth of the parasite population during infection.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected with Leishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified. Copyright © 2000 John Wiley & Sons, Ltd.     

Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice

Chitinases of trypanosomatid parasites have been proposed to fulfil various roles in their blood-feeding arthropod vectors but so far none have been directly tested using a molecular approach. We characterized the ability of Leishmania mexicana episomally transfected with LmexCht1 (the L. mexicana chitinase gene) to survive and grow within the permissive sand fly vector, Lutzomyia longipalpis. Compared with control plasmid transfectants, the overexpression of chitinase was found to increase the average number of parasites per sand fly and accelerate the escape of parasites from the peritrophic matrix-enclosed blood meal as revealed by earlier arrival at the stomodeal valve. Such flies also exhibited increased damage to the structure of the stomodeal valve, which may facilitate transmission by regurgitation. When exposed individually to BALB/c mice, those flies with chitinase-overexpressing parasites spent on average 2.4-2.5 times longer in contact with their host during feeding, compared with flies with control infections. Furthermore, the lesions that resulted from these single fly bite infections were both significantly larger and with higher final parasite burdens than controls. These data show that chitinase is a multifunctional virulence factor for L. mexicana which assists its survival in Lu. longipalpis. Specifically, this enzyme enables the parasites to colonize the anterior midgut of the sand fly more quickly, modify the sand fly stomodeal valve and affect its blood feeding, all of which combine to enhance transmission.     

Attraction of the sandfly Lutzomyia longipalpis to possible biomarker compounds from dogs infected with Leishmania infantum

Lutzomyia longipalpis (Diptera: Psychodidae) is the primary vector of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in the Americas. Studies have been carried out to identify new alternatives for monitoring and controlling this sandfly species, particularly with the use of chemical baits. The attractiveness of odours emitted by foxes and alcohols found in some plants has already been demonstrated in laboratory tests with Lu. longipalpis. However, no studies have evaluated the responses of these insects to volatile organic compounds (VOCs) emitted by dogs. The present study was carried out to investigate the effects on Lu. longipalpis of individual and blends of VOCs identified in hair from dogs infected with L. infantum. Effects in male and female Lu. longipalpis were assessed using wind tunnel methodology. Individual compounds including octanal, nonanal, decanal and heptadecane showed capacity for activating and/or attracting male Lu. longipalpis. Only decanal and nonanal showed effects on females. The combination of octanal, decanal and heptadecane increased activation and attraction behaviour in male sandflies, as did the blend of octanal and decanal. These findings indicate that VOCs emitted by dogs may be an interesting source of new attractants of sandflies.     

Exploring sand fly salivary proteins to design multiepitope subunit vaccine to fight against visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by the parasites of Leishmania donovani complex, leads to the death of 20 000 to 40 000 people from 56 affected countries, worldwide. Till date, there is not a single available vaccine candidate to prevent the VL infection, and treatment only relies upon expensive and toxic chemotherapeutic options. Consequently, immunoinformatics approach was applied to design a multiepitope-based subunit vaccine to enhance the humoral as well as cell-mediated immunity. Constructed vaccine candidate was further subjected to evaluation on allergenicity and antigenicity and physiochemical parameters. Later on, disulfide engineering was performed to increase the stability of vaccine construct. Also, molecular docking and molecular dynamics simulation study were performed to check the binding affinity and stability of toll-like receptor-4 to vaccine construct complex. Finally, codon optimization and in silico cloning were performed to ensure the expression of proposed vaccine construct in a microbial expression system.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Leishmania chagasi naturally resistant to nitric oxide isolated from humans and dogs with visceral leishmaniasis in Brazil

Nitric oxide (NO) plays an important role as a leishmanicidal agent in murine macrophages. NO resistant Escherichia coli and Mycobacterium tuberculosis have been associated with poor outcomes of their resulting diseases. NO resistant Leishmania braziliensis has also been identified and exacerbates the clinical course of human leishmaniasis. We report, for the first time, natural resistance of Leishmania chagasi promastigotes to NO. These parasites were isolated from humans and dogs with visceral leishmaniasis. We also demonstrate that this resistance profile was associated with a greater survival capacity and a greater parasite burden in murine macrophages, independent of activation and after activation by IFN-γ and LPS.     

Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes,dogs and sand flies

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.     

The genetic structure of Leishmania infantum populations in Brazil and its possible association with the transmission cycle of visceral leishmaniasis

Leishmania infantum is the etiologic agent of visceral leishmaniasis (VL) in the Americas, Mediterranean basin and West and Central Asia. Although the geographic structure of L. infantum populations from the Old World have been described, few studies have addressed the population structure of this parasite in the Neotropical region. We employed 14 microsatellites to analyze the population structure of the L. infantum strains isolated from humans and dogs from most of the Brazilian states endemic for VL and from Paraguay. The results indicate a low genetic diversity, high inbreeding estimates and a depletion of heterozygotes, which together indicate a predominantly clonal breeding system, but signs of sexual events are also present. Three populations were identified from the clustering analysis, and they were well supported by F statistics inferences and partially corroborated by distance-based. POP1 (111 strains) was observed in all but one endemic area. POP2 (31 strains) is also well-dispersed, but it was the predominant population in Mato Grosso (MT). POP3 (31 strains) was less dispersed, and it was observed primarily in Mato Grosso do Sul (MS). Strains originated from an outbreak of canine VL in Southern Brazil were grouped in POP1 with those from Paraguay, which corroborates the hypothesis of dispersal from Northeastern Argentina and Paraguay. The distribution of VL in MS seems to follow the west-east construction of the Bolivia-Brazil pipeline from Corumbá municipality. This may have resulted in a strong association of POP3 and Lutzomyia cruzi, which is the main VL vector in Corumbá, and a dispersion of this population in this region that was shaped by human interference. This vector also occurs in MT and may influence the structure of POP2. This paper presents significant advances in the understanding of the population structure of L. infantum in Brazil and its association with eco-epidemiological aspects of VL.     

Mark-release-recapture of sand flies fed on leishmanial dogs: the natural life-cycle of Leishmania infantum in Phlebotomus ariasi

Wild-caught Phlebotomus ariasi Tonnoir permitted to feed on dogs infected with Leishmania infantum Nicolle were marked with fluorescent powder and released into their natural habitat in an uninhabited area of the Cévennes in southern France. Over a period of 29 days after release, 253 females were recaptured with CDC miniature light traps or by active search at night with portable UV lamps. The ovaries and infections in the alimentary tract were then examined. The females oviposited 6 nights after in infecting blood meal. Second blood meals were never taken during the maturation of eggs. During the first ovarian cycle, midgut infections with promastigotes were only moderately heavy. The intensity of infection increased markedly during the second ovarian cycle and, in the third ovarian cycle, the first pharynx infected with paramastigotes was seen (on day 19). From day 19 to day 29, 76% of the flies had pharyngeal infections. Three out of 19 sand flies with pharyngeal infections recaptured during this period had metacyclic promastigotes in their mouthparts. The long time required for parasites to reach the proboscis in completely natural conditions suggests that their presence in the mouthparts is not a prerequisite for transmission by bite. It is more likely that transmission is most commonly by the regurgitation of metacyclic promastigotes from the thoracic midgut following damage to the stomodaeal valve by chitinase produced by the parasite during its development in the gut of the fly. Nevertheless, it is reasonable to assume that the bite of a fly with metacyclic promastigotes in the proboscis (or salivary glands) would also be infective.