A method for the purification of Leishmania promastigotes from infected phlebotomine sandflies

We describe a method for the purification of Leishmania promastigotes, isolated from infected sandflies (Lutzomyia longipalpis) using a discontinuous density centrifugation gradient (Percoll/Homem). The sandflies, infected seven days previously with Leishmania donovani chagasi or Leishmania mexicana mexicana from culture, were homogenized and centrifuged on a Percoll discontinuous gradient. Five interface bands were formed, and most of the promastigotes settled out at the interface between the (30% and 40%) Percoll concentrations. An extraction of 3.5 x 10(4) promastigotes from 90 female flies was achieved using this technique.     

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).     

Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies

The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.     

A brief survey of phlebotomine sandflies in Thailand

Sandflies were found in widely scattered localities in Thailand in varied numbers. Daytime resting places for the adults included caves, termite hills, abandoned houses, ancient stone sanctuaries, air-raid shelters, tree hollows, loose bark of dead standing trees and rock crevices. Of fifteen species, P. major major, P. teshi, S. anodontis, S. gemmea, S. hodgsoni hodgsoni, S. perturbans, S. punjabensis and N. vietnamensis were here recorded for the first time in Thailand. P. argentipes and P. major major are interesting in view of their potential as disease vectors.     

甘肃省的白蛉区系

何观清于 194 8年首次记载了甘肃省的蛉种及其分布的一些资料 ,指出在永靖、临夏、皋兰、通渭、平凉和泾川 6县有中华白蛉的分布 ,在皋兰和永靖还查见了蒙古白蛉[1] 。 195 1年 ,甘肃省建立了黑热病防治所 ,在开展对该病防治工作的同时 ,也对白蛉的种属及其地理分布进行调查。此后 ,虽然分管黑热病 (内脏利什曼病 )防治工作的单位几经更迭 ,但对蛉的调查工作迄未间断 ,除甘南藏族自治州的西部高原地带尚待调查外 ,其它地带 ,包括陇中、陇东黄土高原、陇南山地和河西走廊荒漠 ,都曾先后用人工捕集、捕蛉器和粘性纸等方法收集白蛉并进行鉴定。…     

Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper

The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.     

Real-time PCR assay for the identification of cutaneous Leishmania parasite species in Constantine region of Algeria

We evaluated the use of real-time PCR for the identification of cutaneous Leishmania species. The assay, based on the melting curve analysis of fluorescent products, allows the discrimination of four culture adapted strains (L. infantum MHOM/TN/80/IPT1, L. major MHOM/SU/73/5-ASKH, L. donovani MHOM/IN/80/DD8 and L. tropica MHOM/SU/74/K27). One hundred and twenty-nine skin lesions, spotted on filter paper, were collected from patients consulting for suspicion of cutaneous leishmaniasis (CL) at the parasitology laboratory of Constantine Hospital (Algeria). Ninety-seven (75.2%) of the samples analyzed were positive. Sixty-one (5%) were related to L. major strain. These results indicate that PCR assay provides pleasant results with filter paper and represents a tool for the identification of old world CL.     

基于凝集素基因对部分隐孢子虫种类的分子鉴定

目的 为了解隐孢子虫凝集素基因在不同分离株的序列差异及其遗传进化关系,以SSU rRNA基因作参照,评价Lectin基因位点作为隐孢子虫基因分型和进化关系分析的可行性。方法 采用巢式PCR,分别在Lectin和SSU rRNA位点处对本实验室分离保存的多个隐孢子虫分离株进行PCR的扩增。用Clustalx对扩增序列与参考序列进行比对,用MEGA5中的邻近法(Neighbor joining method NJ),进行进化树的构建。结果 基于Lectin基因位点,在C. parvum、C. hominis、C. cuniculus及其在SSU rRNA 处与人源C. hominis极为相近的horse genotype、驴源C. hominis均成功扩增出了大小在450 bp左右的目的 条带,并进行了进化树的分析,不同种类和同种不同动物来源的隐孢子虫分布在不同的分支上。结论 Lectin基因位点可很好的区分SSU rRNA序列极为相似的几种隐孢子虫,有望为人兽共患隐孢子虫分类和遗传研究提供新的基因靶标。     

Rapid identification of Leishmania complexes by a real-time PCR assay

A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephosphate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identification. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians.     

中国两种钩虫成虫的PCR虫种鉴定研究

目的 对中国流行的美洲钩虫和十二指肠钩虫进行PCR鉴别.方法 通过对中国五省钩虫患者使用双羟萘酸噻嘧啶驱虫获得钩虫成虫,抽提单条美洲钩虫和十二指肠钩虫总DNA(各25条),用美洲钩虫和十二指肠钩虫线粒体DNA细胞色素氧化酶亚基1(mitochondrial cytochrome oxidase 1,CO1)基因特异性引物(NaF-NaR,AdF-AdR)进行PCR扩增.对PCR产物进行电泳、测序.使用相同引物对日本血吸虫、鞭虫、犬钩虫DNA进行PCR扩增.结果 25份美洲钩虫和25份十二指肠钩虫均能各扩增出约500 bp和700 bp的条带,2种PCR产物分别与美洲钩虫CO1(GenBank登录号为AF303136.1)和十二指肠钩虫CO1(GenBank登录号为AJ417718.1)基因片段序列一致性为99％和98％.但2对引物用于其他虫种DNA则无条带.结论 引物NaF-NaR、AdF-AdR能够用于区分在中国流行的美洲钩虫和十二指肠钩虫.     

Identification and differentiation of Iranian Leishmania species by PCR amplification of kDNA

We describe the specific identification of Leishmania species in Iran using PCR DNA amplification of kDNA. For this purpose, we designed a pair of primers--upstream 5' TCGCAGAACGCCCCTACC 3' and downstream 5'-AGGGGTTGGTGTAAAATAGGC 3'--specific for conserved sequences of kDNA of Leishmania. Using this primer, we identified 3 different amplified fragments from the kDNA of the WHO reference Leishmania species. Two bands at 620 and 850 bp were identified for L. major (MRHO/IR/64/Nadim-1 strain) and only 1 band at 620 bp was identified for L. major (P strain). Therefore, we could differentiate 2 Leishmania species. Also, 1 band at 830 bp was identified for L. tropica (MHOM/Sudan/58/OD strain). We determined the sequence analysis of 2 DNA bands (620 and 850 bp) obtained from kDNA of L. major (MRHO/IR/64/Nadim-1). A total of 157 bp from the 5' site and 234 bp from the 3' site were sequenced and showed about 28% homology between 620 and 850 bp fragments. This technique could amplify as little as 1 fg of DNA and was used to differentiate kDNA samples isolated from Iranian patients with cutaneous leishmaniasis. These data indicate that the primer used for PCR amplification of kDNA is specific and can be used for diagnostic and epidemiological purposes.     

Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina

ABSTRACT: BACKGROUND: The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. METHODS: A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical exam, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions. RESULTS: Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis. CONCLUSIONS: The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the specie of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.     

Bionomics of phlebotomine sandflies at a peacekeeping duty site in the north of Sinai, Egypt

A longitudinal entomological survey for sandflies was conducted from 1989 to 1991 at a focus of enzootic cutaneous leishmaniasis in Northeast Sinai, Egypt, within the border region monitored by multinational peacekeepers. Standardized sampling with CDC light traps, oiled paper "sticky traps", and human landing collection was employed to determine monthly trends in species composition, density, sex ratio, and reproductive status of vector sandflies. Each collection method independently defined sandfly seasonality as the period May-November in 1990, and March-October in 1991. Plebotomus papatasi was the only anthropophagic species found and comprised more than 94% of the sandfly population. Two population peaks (May, July) were observed for this species in both survey years. Density of P. papatasi in underground bunkers was higher than outside but inflated by a greater proportion of male flies. During 1990, the proportion of gravid P. papatasi increased progressively during the 5 months period from May to September and averaged 29.5% and 29.7% for interior and exterior collections, respectively. Density of P. papatasi was greater during 1991, but proportions of gravid flies were significantly lower in each survey month and averaged 14.9% and 12.3% for interior and exterior collections, respectively. Seasonal rates of Leishmania-infected P. papatasi averaged 0.8% and 0.9% in 1989 and 1990, but fell to zero in 1991, suggesting an unstable focus of Leishmania major transmission. Proportions of gravid flies may be a valid indicator of the physiological age and epidemiologic importance of the vector sandfly population at this focus. The strong correlation of sticky trap indices to human-landing/biting rates shows that this is an accurate, inexpensive, and no-risk alternative to human bait collections.     

The genital atrium as a good taxonomic character to distinguish between species of phlebotomine sandflies (Diptera: Psychodidae) from Venezuela

The shape and size of the genital atria of 17 phlebotomine sandflies from Venezuela were examined. The atria were found to express constant characters among individuals of the same species and to be sufficiently different between species to allow taxonomic separation. The spines on the genital armature are described and the characters that can be used to classify individual specimens to species level identified. It is suggested that these characters can be of use on specimens where the spermathecae have been lost or where cryptic species are concerned. Dissection procedures to display the atrium are given.