First Evidence of a Hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana DNA Detected from the Phlebotomine Sand Fly Lutzomyia tejadai in Peru

The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area.     

Molecular Detection of Leishmania in Phlebotomine Sand Flies (Diptera: Psychodidae) from a Cutaneous Leishmaniasis Focus at Xakriabá Indigenous Reserve,Brazil

Autochthonous cases of American cutaneous leishmaniasis (ACL) have been reported since 2001 in the Xakriabá Indigenous Reserve located in the municipality of São João das Missões in northern Minas Gerais state, Brazil. In order to study the presence of Leishmania DNA in phlebotomine sand flies, six entomological collections were carried out from July 2008 through July 2009, using 40 light traps placed in peridomicile areas of 20 randomly selected houses. From October 2011 through August 2012, another six collections were carried out with 20 light traps distributed among four trails (five traps per trail) selected for a previous study of wild and synanthropic hosts of Leishmania. A total of 4,760 phlebotomine specimens were collected belonging to ten genera and twenty-three species. Single female specimens or pools with up to ten specimens of the same locality, species and date, for Leishmania detection by molecular methods. Species identification of parasites was performed with ITS1 PCR-RFLP using HaeIII enzyme and genetic sequencing for SSU rRNA target. The presence of Leishmania DNA was detected in eleven samples from peridomicile areas: Lu. longipalpis (two), Nyssomyia intermedia (four), Lu. renei (two), Lu. ischnacantha, Micropygomyia goiana and Evandromyia lenti (one pool of each specie). The presence of Leishmania DNA was detected in twelve samples from among the trails: Martinsmyia minasensis (six), Ny. intermedia (three), Mi. peresi (two) and Ev. lenti (one). The presence of Leishmania infantum DNA in Lu. longipalpis and Leishmania braziliensis DNA in Ny. intermediasupport the epidemiological importance of these species of sand flies in the cycle of visceral and cutaneous leishmaniasis, respectively. The results also found other species associated with Leishmania DNA, such as Mt. minasensis and Ev. lenti, which may participate in a wild and/or synanthropic cycle of Leishmania transmission in the studied area.     

Tabanid Flies (Diptera: Tabanidae) of the Northern Part of Gyeonggi Province, Republic of Korea

Tabanid flies were surveyed using New Jersey light traps and Mosquito Magnet® traps at US Army installations and training sites in the northern part of Gyeonggi Province, Republic of Korea. A total of 344 tabanid flies, comprising 14 species and 5 genera, were collected from June to August during 2003‐2004. Dominant species were Chrysops vanderwulpi (57.3%), Haematopota sinensis (29.1%), Haematopota koryoensis (4.4%) and Tabanus mandarinus (2.3%). Mosquito Magnet traps that generated CO₂through the combustion of propane gas were nearly 6 times more effective in collecting tabanid flies than New Jersey light traps. Based on Mosquito Magnet trap collections, peak populations occurred from mid‐June to early July.;     

Comparative efficacy of three suction traps for collecting phlebotomine sand flies (Diptera: Psychodidae) in open habitats

The efficacy of three suction traps for trapping phlebotomine sand flies (Diptera: Psychodidae) was compared. Traps were baited with Co₂ and used without any light source. CO₂‐baited CDC traps were evaluated either in their standard downdraft orientation or inverted (iCDC traps). Mosquito Magnet‐X (MMX) counterflow geometry traps were tested in the updraft orientation only. Both updraft traps (iCDC and MMX) were deployed with their opening ～10 cm from the ground while the opening of the downdraft (CDC) trap was ～40 cm above ground. Comparisons were conducted in two arid locations where different sand fly species prevail. In the Jordan Valley, 3,367 sand flies were caught, 2,370 of which were females. The predominant species was Phlebotomus (Phlebotomus) papatasi, Scopoli 1786 (>99%). The updraft‐type traps iCDC and MMX caught an average of 118 and 67.1 sand flies per trap night, respectively. The CDC trap caught 32.9 sand flies on average per night, significantly less than the iCDC traps. In the Judean desert, traps were arranged in a 3×3 Latin square design. A total of 565 sand flies were caught, 345 of which were females. The predominant species was P. (Paraphlebotomus) sergenti Parrot 1917 (87%). The updraft traps iCDC and MMX caught an average of 25.6 and 17.9 sand flies per trap per night, respectively. The CDC trap caught 7.8 sand flies on average per night, significantly less than the iCDC traps. The female to male ratio was 1.7 on average for all trap types. In conclusion, updraft traps deployed with their opening close to the ground are clearly more effective for trapping sand flies than downdraft CDC traps in open habitats.     

Richness and diversity of sand flies (Diptera,Psychodidae) in an Atlantic rainforest reserve in southeastern Brazil

Our objective was to study and evaluate the richness and diversity of Phlebotominae fauna in the Duas Bocas Biological Reserve (DBBR) in the state of Espírito Santo, in southeastern Brazil. Sand fly collections were carried out during four consecutive nights each month between August 2007 and July 2008 at DBBR by using CDC automatic light traps and an illuminated Shannon trap. Specific richness (S) and Shannon diversity index (H) was calculated for each trap. We collected 18,868 sand flies belonging to 29 species and 13 genera. Nyssomyia yuilli yuilli was the most abundant species followed by Psychodopygus ayrozai, Ps. hirsutus, Psathyromyia pascalei, and Ps. matosi. We recorded Brumptomyia cardosoi, Br. troglodytes, and Ps. geniculatus for the first time in the state of Espírito Santo. We discuss the differences in diversity and richness of the sand flies in both traps and in relation to other Brazilian localities and biomes. We also discuss the possibility of wild transmission of Leishmania in the DBBR and the influence of the sand fly species in leishmaniasis transmission to the adjacent areas of the reserve.     

Orientation of Culex mosquitoes to carbon dioxide-baited traps: flight manoeuvres and trapping efficiency

Females of Culex quinquefasciatus Say and Culex tarsalis Coquillet (Diptera: Culicidae) in the host-seeking stage were released and video recorded in three dimensions in a large field wind tunnel as they flew to four kinds of CO2-baited mosquito traps. The trapping efficiency (number of mosquitoes approaching compared to the number caught) was determined for each trap type. The Encephalitis Virus Surveillance (EVS), Mosquito Magnet Freedom (MMF) and Mosquito Magnet Liberty (MML) traps captured only 13-16% of approaching Cx. quinquefasciatus females, whereas the Mosquito Magnet-X (MMX) trap captured 58%. Similar results were obtained for Cx. tarsalis. Orientation behaviour and flight parameters of mosquitoes approaching the four traps were compared. Mosquitoes spent the most time orienting to the EVS trap. Flight speed decreased as mosquitoes entered the vicinity of each trap and a large portion of their time was spent within 30 cm downwind of the traps. Flights became highly tortuous downwind of the poorly performing traps and just upwind of the MMX trap. Differences between traps and possible explanations for the superior performance of the MMX trap are considered.     

Comparison of plume structures of carbon dioxide emitted from different mosquito traps

A large field wind tunnel was used to compare four types of CO2-baited mosquito traps. This study compared the plume structure and concentration of CO2 emitted by each trap, flow of suction into the trapping systems, flow of CO2 being released, trap shape and configuration, differences in visual appearance, and differences in temperature and humidity of emissions at the source of CO2 compared to ambient air. The structure of the CO2 plumes emitted by each trap differed considerably. All four plumes were turbulent, causing the concentration of CO2 within several metres of the source to attenuate to between 375 and 875 p.p.m. The Encephalitis Virus Surveillance (EVS) trap emitted concentrations of CO2 exceeding 20,000 p.p.m., the detection limits of our equipment, whereas the Mosquito Magnet Freedom (MMF), Mosquito Magnet Liberty (MML) and Mosquito Magnet X (MMX) traps released CO2 at peaks of about 3500, 7200 and 8700 p.p.m., respectively. The MMX trap produced the greatest air velocity at both the suction inlet and CO2 outlet, followed by the MMF, MML and the EVS traps, respectively.     

Lutzomyia Sand Fly Diversity and Rates of Infection by Wolbachia and an Exotic Leishmania Species on Barro Colorado Island,Panama

Background

Sand flies (Diptera, Psychodidae, Phlebotominae) in the genus Lutzomyia are the predominant vectors of the protozoan disease leishmaniasis in the New World. Within the watershed of the Panama Canal, the cutaneous form of leishmaniasis is a continuous health threat for residents, tourists and members of an international research community. Here we report the results of screening a tropical forest assemblage of sand fly species for infection by both Leishmania and a microbe that can potentially serve in vector population control, the cytoplasmically transmitted rickettsia, Wolbachia pipientis. Knowing accurately which Lutzomyia species are present, what their evolutionary relationships are, and how they are infected by strains of both Leishmania and Wolbachia is of critical value for building strategies to mitigate the impact of this disease in humans.

Methodology and Findings

We collected, sorted and then used DNA sequences to determine the diversity and probable phylogenetic relationships of the Phlebotominae occurring in the understory of Barro Colorado Island in the Republic of Panama. Sequence from CO1, the DNA barcoding gene, supported 18 morphology-based species determinations while revealing the presence of two possible “cryptic” species, one (Lu. sp. nr vespertilionis) within the Vespertilionis group, the other (Lu. gomezi) within the Lutzomyia-cruciata series. Using ITS-1 and “minicircle” primers we detected Leishmania DNA in 43.3% of Lu. trapidoi, 26.3% of Lu. gomezi individuals and in 0% of the other 18 sand fly species. Identical ITS-1 sequence was obtained from the Leishmania infecting Lu. trapidoi and Lu. gomezi, sequence which was 93% similar to Leishmania (viannia) naiffi in GenBank, a species previously unknown in Panama, but recognized as a type of cutaneous leishmaniasis vectored broadly across northern and central South America. Distinct strains of the intracellular bacterium Wolbachia were detected in three of 20 sand fly species, including Lu. trapidoi, in which it frequently co-occurred with Leishmania.

Conclusions

Both morphological and molecular methods were used to examine an assemblage of 20 sand fly species occurring in the forests of the Panama Canal area. Two of these species, members of separate clades, were found to carry Leishmania at high frequency and hence are likely vectors of leishmaniasis to humans or other mammal species. A single Leishmania species, identified with high confidence as Le. naiffi, was carried by both species. That Le. naiffi is known to cause cutaneous lesions in South America but has hitherto not been reported or implicated in Panama opens the possibility that its range has recently expanded to include the Isthmus or that it occurs as a recent introduction. The occurrence of Leishmania and Wolbachia in Lu. trapidoi identifies one important vector of the disease as a potential target for gene introductions using Wolbachia population sweeps.     

Evaluating the phototactic behavior responses of the diamondback moth,Plutella xylostella,to some different wavelength LED lights in laboratory and field

Light traps equipped with light emitting diodes (LEDs) have been applied to manage some phototactic insect pests. The diamondback moth, Plutella xylostella, is a cosmopolitan insect pest to be seriously harmful to many cruciferous plants. The present research focused on evaluating the phototactic behavior responses of the moths to several wavelengths and photon flux densities of LED lights under laboratory and field conditions. The results from the laboratory showed that the highest phototactic behavior responses of P. xylostella moths were recorded for UV (380 nm) LED light under 1.5 µmol m⁻² s⁻¹ and 2.5 µmol m⁻² s⁻¹. The moths were more attracted to light traps equipped with 4 LEDs and 6 LEDs of 380 nm, respectively, between 20:00 and 22:00 than the other groups and night times in the field. The finding from the field was consistent with the results from the laboratory. We found that the 380 nm LED light results in the strongest attraction rate of the moths by 92.4 % and the moths caught in light trap with the UV LEDs was 1.62 times more than that with a black light. These data clearly demonstrate that P. xylostella moths have a high sensitivity to 380 nm, therefore, a 380 nm LED light trap could be useful for monitoring and controlling the moths.     

Leishmania (Viannia) naiffi: rare enough to be neglected?

In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and presents a wide spectrum of clinical manifestations due, in part, to the circulation of at least seven Leishmania species. Few reports of Leishmania (Viannia) naiffi infection suggest that its occurrence is uncommon and the reported cases present a benign clinical course and a good response to treatment. This study aimed to strengthen the clinical and epidemiological importance of L. (V.) naiffi in the Amazon Region (Manaus, state of Amazonas) and to report therapeutic failure in patients infected with this species. Thirty Leishmania spp samples isolated from cutaneous lesions were characterised by multilocus enzyme electrophoresis. As expected, the most common species was Leishmania (V.) guyanensis (20 cases). However, a relevant number ofL. (V.) naiffi patients (8 cases) was observed, thus demonstrating that this species is not uncommon in the region. No patient infected withL. (V.) naiffi evolved to spontaneous cure until the start of treatment, which indicated that this species may not have a self-limiting nature. In addition, two of the patients experienced a poor response to antimonial or pentamidine therapy. Thus, either ATL cases due to L. (V.) naiffi cannot be as uncommon as previously thought or this species is currently expanding in this region.     

Sandflies (Diptera: Psychodidae) in rural and urban environments in an endemic area of cutaneous leishmaniasis in southern Brazil

The high proportion of cases of cutaneous leishmaniasis reported amongst residents in the city of Bandeirantes, in the state of Paraná, Brazil, led the authors to investigate the phlebotomine fauna in both urban and rural environments. The sandflies were captured with automatic light traps from 07:00 pm-07:00 am fortnightly in 11 urban peridomiciles from April 2008-March 2009 and monthly in three ecotopes within four rural localities from April 2009-March 2010. In one of these latter localities, sandfly capture was conducted with white/black Shannon traps during each of three seasons: spring, summer and fall. A total of 5,729 sandflies of 17 species were captured. Nyssomyia neivai (46.7%) and Nyssomyia whitmani (35.3%) were the predominant species. In this study, 3,865 specimens were captured with automatic light traps: 22 (0.083 sandflies/trap) in the urban areas and 3,843 (26.69 sandflies/trap) in the rural areas. Ny. neivai was predominant in urban (68.2%) and rural (42.8%) areas. A total of 1,864 specimens were captured with the white/black Shannon traps and Ny. neivai (54.5%) and Ny. whitmani (31.4%) were the predominant species captured. The small numbers of sandflies captured in the urban areas suggest that the transmission of Leishmania has occurred in the rural area due to Ny. neivai and Ny. whitmani as the probable vectors.     

Colonization of Lutzomyia verrucarum and Lutzomyia longipalpis Sand Flies (Diptera: Psychodidae) by Bartonella bacilliformis,the Etiologic Agent of Carrión’s Disease

Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión’s disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.     

The Distribution and Diversity of Bartonella Species in Rodents and Their Ectoparasites across Thailand

Our study highlights the surveillance of Bartonella species among rodents and their associated ectoparasites (ticks, fleas, lice, and mites) in several regions across Thailand. A total of 619 rodents and 554 pooled ectoparasites (287 mite pools, 62 flea pools, 35 louse pools, and 170 tick pools) were collected from 8 provinces within 4 regions of Thailand. Bandicota indica (279), Rattus rattus (163), and R. exulans (96) were the most prevalent species of rats collected in this study. Real-time PCR assay targeting Bartonella-specific ssrA gene was used for screening and each positive sample was confirmed by PCR using nuoG gene. The prevalence of Bartonella DNA in rodent (around 17%) was recorded in all regions. The highest prevalence of Bartonella species was found in B. savilei and R. rattus with the rate of 35.7% (5/14) and 32.5% (53/163), respectively. High prevalence of Bartonella-positive rodent was also found in B. indica (15.1%, 42/279), and R. norvegicus (12.5%, 5/40). In contrast, the prevalence of Bartonella species in ectoparasites collected from the rats varied significantly according to types of ectoparasites. A high prevalence of Bartonella DNA was found in louse pools (Polyplax spp. and Hoplopleura spp., 57.1%) and flea pools (Xenopsylla cheopis, 25.8%), while a low prevalence was found in pools of mites (Leptotrombidium spp. and Ascoschoengastia spp., 1.7%) and ticks (Haemaphysalis spp., 3.5%). Prevalence of Bartonella DNA in ectoparasites collected from Bartonella-positive rodents (19.4%) was significantly higher comparing to ectoparasites from Bartonella-negative rodents (8.7%). The phylogenetic analysis of 41 gltA sequences of 16 Bartonella isolates from rodent blood and 25 Bartonella-positive ectoparasites revealed a wide range of diversity among Bartonella species with a majority of sequences (61.0%) belonging to Bartonella elizabethae complex (11 rodents, 1 mite pool, and 5 louse pools), while the remaining sequences were identical to B. phoceensis (17.1%, 1 mite pool, 5 louse pools, and 1 tick pool), B. coopersplainensis (19.5%, 5 rodents, 1 louse pool, and 2 tick pools), and one previously unidentified Bartonella species (2.4%, 1 louse pool).     

Development of a System for Genetic Manipulation of Bartonella bacilliformis

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan^r clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.     

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.     

Response of phlebotomine sand flies to light-emitting diode-modified light traps in southern Egypt.

Centers For Disease Control and Prevention (CDC) light traps were modified for use with light-emitting diodes (LED) and compared against a control trap (incandescent light) to determine the effectiveness of blue, green, and red lights against standard incandescent light routinely used for sand fly surveillance. Light traps were baited with dry ice and rotated through a 4 x 4 Latin square design during May, June, and July, 2006. Trapping over 12 trap nights yielded a total of 2,298 sand flies in the village of Bahrif, 6 km north of Aswan on the east bank of the Nile River in southern Egypt. Phlebotomus papatasi comprised 94.4% of trap collections with five other species collected in small numbers. Over half (55.13%) of all sand flies were collected from red light traps and significantly more sand flies (P < 0.05) were collected from red light traps than from blue, green, or incandescent light traps. Red light traps collected more than twice as many sand flies as control (incandescent) traps and > 4 x more than blue and green light traps. Results indicate that LED red light is a more effective substitute for standard incandescent light when surveying in areas where P. papatasi is the predominant sand fly species. Each LED uses approximately 15% of the energy that a standard CDC lamp consumes, extending battery life and effective operating time of traps. Our prototype LED-modified traps performed well in this hot, arid environment with no trap failures.     

Aging of Utricularia traps and variability of microorganisms associated with that microhabitat

Various authors have described algae in aquatic Utricularia traps as commensals, as stress factors or as prey. This study examined the diversity and abundance of organisms (prey, algae, protozoa and bacteria) in the traps of aquatic Utricularia reflexa in relation to prey occurrence and trap age. The number of organisms increased with the trap age. In both young and old traps, phytoplankton dominated of all organisms found. In young traps, Scenedesmus spp. and Characiopsis sp. were the most abundant algae, while Scenedesmus spp. and the palmelloidal form of Euglena spp. dominated in the old traps. Most of the algal species found stayed alive in the trap environment. The number of living algae and ciliates inside the traps increased with the increasing trap age, too. As the number of Paramecium bursaria inside traps consistently increased with the trap age and number of bacteria, which serve as a food for them, ciliates can be regarded as commensals, but not as prey for the plant. The predominant organisms in the traps were those that can be considered either commensals or intruders, exceeding captured macroscopic prey.     

Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis

BackgroundPhlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs.ConclusionsThese results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.     

Rapid and Sensitive Detection of Bartonella bacilliformis in Experimentally Infected Sand Flies by Loop-Mediated Isothermal Amplification (LAMP) of the Pap31 Gene

Background

Carrion'' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis.

Methods and Findings

The sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis.

Conclusions

The LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector.