Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.     

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.     

Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina

The natural infection of Lutzomyia neivai with Leishmania in the endemic area of American cutaneous leishmaniasis (ACL) in northwestern Argentina was analyzed by the polymerase chain reaction (PCR)-hybridization technique. Phlebotominae sand flies were captured in the provinces of Tucumán and Salta between 1999 and 2003. From a sample of 440 Lu. neivai females analysed for the detection of the Leishmania (Viannia) and Leishmania (Leishmania) subgenera, 9.1% of the samples resulted infected with a parasite of the subgenus Viannia and none with the Leishmania. This is the first report of naturally infected sand flies in Argentina besides the first report of infected Lu. neivai sensu strictu. Our results contributed to further incrimination of this specie as vector of leishmaniasis in the area and the identification of the main circulating parasite as belonging to the Leishmania (Viannia) subgenera.     

Breeding sites of phlebotomine sand flies (Diptera: Psychodidae) and efficiency of extraction techniques for immature stages in terra-firme forest in Amazonas State, Brazil

Information on natural breeding sites of phlebotomine sand flies is scanty, due to the difficulties of isolation of immatures from the soil where they occur. The present study investigated breeding sites in several microhabitats in a “terra-firme” forest in Pitinga, Amazonas State, Brazil. Results on the efficacy of different extraction techniques used for isolating sand flies, and the temperature and the pH of the samples collected, are presented. Samples of soil and organic matter from different microhabitats, processed by floatation-sieving, direct examination, Berlese–Tullgren, and emergence cages, revealed, for the first time in Amazonas, breeding sites in five microhabitats (tree bases, unsheltered forest floor, soil from under fallen logs, soil from under roots, and palm-tree bases). Overall, 138 immatures and 29 newly emerged adults were recovered from these microhabitats. The abundance of immatures in samples close to tree bases was significantly higher than in more open sites not adjacent to tree bases. Floatation-sieving and direct examination were the most effective techniques for immature extraction and survival, respectively. Eleven species of the genus Lutzomyia s.l. were identified, with Lutzomyia monstruosa (Floch & Abonnenc) and Lutzomyia georgii Freitas & Barrett being the most abundant. Differences in the specific composition and relative abundance of the immature and adult sand flies on tree bases suggest that breeding sites may be distant from resting or aggregation sites of adults. The pH, which revealed a slightly acidic soil, as well as the temperature, did not show any significant correlation with the number of immature sand flies collected.     

Canine visceral leishmaniasis in Teresina, Brazil: Relationship between clinical features and infectivity for sand flies

Leishmania chagasi is an intracellular parasite transmitted by the bite of the phlebotomine sand fly Lutzomyia longipalpis, which is the most important of American visceral leishmaniasis. In the gut of the vector, amastigoste forms of the parasite transform into metacyclic promastigotes, from there to the foregut, where they could be transmitted in the next blood meal. Xenodiagnosis is an important tool for the detection of Leishmania, especially when associated to molecular techniques, both being useful for the monitoring and evaluation of dog infectivity in endemic areas. In this study, direct search of Leishmania from material obtained through xenodiagnosis performed in dogs captured in Teresina (Piauí State, Brazil) identified that the predominant forms of the parasite were the procyclic and metacyclic forms located in the hindgut, detected between the 5th and 6th day after the blood meal. Using polymerase chain reaction (PCR), we revealed that dogs with different clinical status were able to infect phlebotomines, the rates of sand fly infection being higher for symptomatic dogs (13%) as compared to asymptomatic ones (3.5%). The direct search was able to demonstrate infection only in phlebotomines in which the blood meal was performed on symptomatic dogs, with a rate of infection of 1.6%. The results underline the importance of using PCR and xenodiagnosis for the detection of Leishmania sp. And for the evaluation of infectivity of dogs in endemic areas, especially those that are asymptomatic.     

Two remarkable pinworms (Nematoda: Enterobiinae) parasitizing orangutan (Pongo abelii) in the Sumatra (Indonesia) including Lemuricola (Protenterobius) pongoi n.sp.

Summary  Two species of pinworms (Enterobiinae) were collected from fresh faeces of semi-wild orangutans Pongo abelii Lesson living in northern Sumatra (Indonesia). The female of Enterobius (Enterobius) buckleyi Sandosham, 1950 is redescribed. Lemuricola (Protenterobius) pongoi n. sp. is described on the basis of females (no males are available) and distinguished from L. (P.) nycticebi (Baylis, 1928) by cephalic and mouth morphology (head and teeth superstructures), body (9.85–15.46 mm) and tail (2.34–2.95 mm) length, smaller eggs (48–56 x 22–28 μm), longer vulva distance from anterior extremity (2.05–3.09 mm) and other features. Characteristic is the total body length/oesophagus length ratio (1: 15.3–22.0). Both nematode species were studied using scanning electron microscopy for the first time.     

Survey of Wild Mammal Hosts of Cutaneous Leishmaniasis Parasites in Panamá and Costa Rica

The eco-epidemiology of American cutaneous leishmaniasis (ACL) is driven by animal reservoir species that are a source of infection for sand flies that serve as vectors infecting humans with Leishmania spp parasites. The emergence and re-emergence of this disease across Latin America calls for further studies to identify reservoir species associated with enzootic transmission. Here, we present results from a survey of 52 individuals from 13 wild mammal species at endemic sites in Costa Rica and Panama where ACL mammal hosts have not been previously studied. For Leishmania spp. diagnostics we employed a novel PCR technique using blood samples collected on filter paper. We only found Leishmania spp parasites in one host, the two-toed sloth, Choloepus hoffmanni. Our findings add further support to the role of two-toed sloths as an important ACL reservoir in Central America.     

Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass (Dicentrarchus labrax)

The present study addresses the role of aromatase and estrogen receptors in sex differentiation and development. With this purpose, a sea bass female- and a male-dominant group were obtained by successive size gradings since in this species females are already larger than males at the time of sex differentiation. Changes in cyp19a and cyp19b gene expression and enzymatic activity were monitored by a validated real-time PCR and a tritiated water assay, respectively, during early development and sex differentiation. Changes in mRNA expression of estrogen receptors, both erb1 and erb2, were also assessed during this period. Results show clear sex-related differences in cyp19a gene expression and enzymatic activity in gonads, with females exhibiting significantly higher levels than males at 150 days post hatching (DPH), when histological signs of sex differentiation were evident. cyp19b gene expression and activity in brain were detectable during early ontogenesis at 50 DPH but no clear sex-related differences were observed. Both erb1 and erb2 showed higher gene expression levels in testis than in ovaries around 200–250 DPH, corresponding with the time of testicular differentiation and precocious male maturation, but no sex-related differences were found in the brain. Together these results indicate that in the European sea bass high expression levels of cyp19a are associated with ovarian differentiation and thus cyp19a can be considered as a suitable molecular marker of ovarian differentiation. However, the involvement of cyp19b in sex differentiation cannot be concluded. In addition, the higher levels of erb1 and erb2 in males versus females during sex differentiation, coinciding with precocious male maturation in the sea bass, suggest an important role for these receptors in testicular development and maturation.     

Carcinogenicity and organ specificity of N-trimethylsilylmethyl-N-nitrosourea (TMS-MNU), N-neopentyl-N-nitrosourea (neoPNU), and N-methyl-N-nitrosourea (MNU) in rats

Summary The carcinogenicity and organ specificity of TMS-MNU and neoPNU, a carbon-analogue of TMS-MNU, in rats were investigated and compared with those of MNU. Compounds were dissolved in olive oil and rats in the experimental groups received 20 weekly intragastric intubations of 10 mg/kg of MNU or equimolar amounts of TMS-MNU or neoPNU in the same manner. The experiment was terminated when the survivors were sacrificed at the 52nd week after the final adminstration. In the TMS-MNU and MNU groups, tumors of the forestomach were induced and the incidence was 100% in the groups of both sexes. In addition, tumors of the glandular stomach, nervous system, kidney, and lung were also observed in these groups. Neurogenic tumors were found more frequently in the MNU group than in the TMS-MNU group. The incidence of lung tumors, however, was higher in the TMS-MNU group than in the MNU group. On the other hand, in the control and neoPNU groups, no tumor was found in these organs except the lung, and all tumors observed in these two groups were histologically similar to spontaneous ones in this strain of rats. These results indicate that the carcinogenicity of N-alkyl-N-nitrosoureas is dependent on the chemical structure of their alkyl chain. The result of the present study coincides with the previous result that the species of TMS-MNU in the alkylating step is the same as that of MNU, but different from neoPNU. The difference in the organ specificity between TMS-MNU and MNU demonstrates that the organ specificity is dominantly dependent on the distribution of the chemicals, since TMS-MNU may possibly be distributed differently from MNU because of its different partition property.Abbreviations TMS-MNU N-trimethylsilylmethyl-N-nitrosourea - neoPNU N-neopentyl-N-nitrosourea - MNU N-methyl-N-nitrosourea Part of this work was presented at the 46th Annual Meeting of the Japanese Cancer Association in Tokyo September, 1987     

Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in São Vicente Férrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.     

Epidemiological aspects of cutaneous leishmaniasis in the Iguazú falls area of Argentina

Over the last three decades the incidence of American cutaneous leishmaniasis (ACL) has increased sharply in Argentina and throughout the world. In the Iguazú Falls area, on the border between Brazil and Paraguay, the incidence of human ACL has risen since 2004. Most of the 36 cases of human ACL reported until 2005 have involved males over 15 years old (75%) infected during deforestation to establish individual farms. Captures carried out in primary forest, periurban areas, and deforested land sites yielded 18,438 sand flies belonging to 13 species; the most prevalent species were Lutzomyia (Nyssomyia) whitmani (87.4%) and Lutzomyia (Mygonemyia.) migonei (7.6%). Cluster analysis was used to group traps according to species and abundance of sand flies. The group of traps located in recently deforested places, in pig and chicken dwellings of houses where ACL cases had been reported in the past, and at one house with an active ACL case, had the highest abundance of Lu. whitmani and Lu. whitmani + Lu. migonei as well as the highest ratio of Lu. whitmani/Lu. migonei. Leishmania sp. infections, both in Lu. whitmani, in Lu. quinquefer, and in smears from human cases were detected by DNA kinetoplast amplification using a generic PCR protocol. The risk of ACL outbreak in the Iguazú Falls area is still associated with economic and leisure activities in primary-secondary forest, including deforestation, rural settlements, fishing, hunting, and ecotourism. In addition, the risk of periurban transmission seems likely, and this is discussed within the framework of surveillance and prevention strategies.     

Morphometrics of females,juveniles and a hermaphrodite of <Emphasis Type="Italic">Longidorus distinctus</Emphasis> Lamberti <Emphasis Type="Italic">et al.</Emphasis>, 1983 (Nematoda: Longidoridae) from Slovakia

Summary A population of Longidorus distinctus Lamberti, Choleva & Agostinelli, 1983 was recovered from soil samples collected from the rhizosphere of plum trees at Král’ovsky Chlmec in south-eastern Slovakia. The area is characterized by sandy soil derived from dune sand. The population comprised females, juveniles of four developmental stages and a bisexual specimen. No males were found. It is the first record of this species from Slovakia. Two females were sequenced and the sequence was deposited in Gen-Bank.     

The first report of authochthonous dirofilariosis in dogs in the Czech Republic

Summary In the Czech Republic, canine dirofilarial infection (Dirofilaria immitis and D. repens) is usually diagnosed in dogs coming from endemic areas and as such has been considered an imported infection. Here, 77 dogs that had never travelled abroad from the Břeclav area, close to Slovak border, were tested for Dirofilaria spp. infection. The presence of microfilaria in peripheral blood was detected by Knott test. Microfilariae were further examined by acid phosphatase staining and molecular methods (PCR). The presence of adult female D. immitis circulating antigens in blood was assessed by a commercial kit (PetChek, IDEXX Laboratories, Portland, USA). Microfilariae were detected in 7 (9 %) out of the 77 animals by the Knott test. The result of the acid phosphatase staining and PCR for all seven samples agreed with Dirofilaria repens species. Other five dogs of the 77 sera (6.5 %) sampled were serologically positive for circulating D. immitis antigens. No D. immitis microfilariae were found in these five dogs. D. repens positive dogs were negative on the ELISA for D. immitis. This is the first report of autochthonous cases of heart-worm disease and subcutaneous dirofilariosis in dogs in the Czech Republic.     

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.     

ASPECTS OF THE ECOLOGY OF PHLEBOTOMINES (Diptera: Psychodidae: Phlebotominae) IN AN AREA OF CUTANEOUS LEISHMANIASIS OCCURRENCE,MUNICIPALITY OF ANGRA DOS REIS,COAST OF RIO DE JANEIRO STATE,BRAZIL

Over a complete two-year period, phlebotomine specimens were caught in an area of cutaneous leishmaniasis occurrence in the municipality of Angra dos Reis. A manual suction tube was used to catch phlebotomines on house walls, and also light traps in domestic and peridomestic settings and in the forest. This yielded 14,170 specimens of 13 species: two in the genus Brumptomyia and eleven in the genus Lutzomyia. L. intermedia predominantly in domestic and peridomestic settings, with little presence in the forest, with the same trend being found in relation to L. migonei, thus proving that these species have adapted to the human environment. L. fischeri appeared to be eclectic regarding location, but was seen to be proportionally more endophilic. L. intermedia and L. migonei were more numerous in peridomestic settings, throughout the year, while L. fischeri was more numerous in domestic settings except in March, April, May and September. From the prevalence of L. intermedia, its proven anthropophily and findings of this species naturally infected with Leishmania (Viannia) braziliensis, it can be incriminated as the main vector for this agent of cutaneous leishmaniasis in the study area, especially in the peridomestic environment. L. fischeri may be a coadjuvant in carrying the parasite.     

Dirofilariosis in Slovakia - a new endemic area in Central Europe

Summary The first systematic research on canine dirofilariosis in Slovakia started in February 2007. In total 287 dogs of various age, sex, breeds, and utilization from southern Slovakia were examined within the study until September 2007. Microfilariaemia was detected in 99 (34.5 %) blood samples. Histochemical staining and PCR approach were used for Dirofilaria species identification. Dirofilaria repens was confirmed in all infected dogs; it occurred in 92 (32.1 %) dogs as a single infection and in 6 (2.1 %) dogs in mixed infection with Dirofilaria immitis. For the first time both species were detected in Eastern Slovakia. Outdoor keeping and age higher 3 years become evident as important risk factors. Male individuals and dogs with long hair were significantly more frequently infected when compared with females and short hair breeds. The present study revealed the occurrence of highly endemic area of dirofilariosis in Slovakia and certified that climatic conditions in the country are favourable for development of the parasite and for further spread of this zoonotic infection.     

First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species

Epidemiological surveillance of leishmaniasis was conducted in a northern Amazonian region of Ecuador, in which cutaneous leishmaniasis cases were recently reported. Sand flies were captured in the military training camp, and the natural infection of sand flies by Leishmania species was examined. Out of 334 female sand flies dissected, the natural infection by flagellates was microscopically detected in 3.9% of Lutzomyia yuilli yuilli and 3.7% of Lutzomyia tortura, and the parasite species were identified as Endotrypanum and Leishmania (Viannia) naiffi, respectively. After the sand fly surveillance, specimens from cutaneous leishmaniasis (CL) patients considered to have acquired the infection in the training camp area were obtained, and the infected parasite species were identified as L. (V.) naiffi. The present study reported first cases of CL caused by L. (V.) naiffi infection in Ecuador. In addition, a high ratio of infection of Lu. tortura by L. (V.) naiffi in the same area strongly suggested that Lu. tortura is responsible for the transmission of L. (V.) naiffi in this area.     

Man-biting sand fly species and natural infection with the Leishmania promastigote in leishmaniasis-endemic areas of Ecuador

A countrywide surveillance of sand flies was performed to obtain information on their geographical distribution and natural infection by Leishmania protozoa in Ecuador. A total of 18,119 sand flies were collected by human landing collections during 32 years from 1982 to 2014, and 29 species were recognized. The most prevalent 10 species were Lutzomyia gomezi, Lu. robusta, Lu. hartmanni, Lu. shannoni, Lu. trapidoi, Lu. panamensis, Lu. maranonensis, Lu. ayacuchensis, Lu. tortura and Lu. yuilli yuilli, and their topographical and vertical distributions were identified. Among all the sand flies, only 197 (1.09%) flies of four Lutzomyia species, Lu. gomezi, Lu. trapidoi, Lu. tortura and Lu. ayacuchensis, were positive for Leishmania. Endotrypanum, a flagellate parasite not pathogenic to humans, were detected in five Lutzomyia species, Lu. robusta, Lu. hartmanni, Lu. trapidoi, Lu. panamensis and Lu. yuilli yuilli, suggesting wide vector-ranges of Endotrypanum species. These data on the genus Lutzomyia and their natural infections with Leishmania and Endotrypanum will be useful for transmission studies and surveillance of leishmaniasis.     

Sand Fly Fauna (Diptera,Pcychodidae, Phlebotominae) in Different Leishmaniasis-Endemic Areas of Ecuador,Surveyed Using a Newly Named Mini-Shannon Trap

To study the sand fly fauna, surveys were performed at four different leishmaniasis-endemic sites in Ecuador from February 2013 to April 2014. A modified and simplified version of the conventional Shannon trap was named “mini-Shannon trap” and put to multiple uses at the different study sites in limited, forested and narrow spaces. The mini-Shannon, CDC light trap and protected human landing method were employed for sand fly collection. The species identification of sand flies was performed mainly based on the morphology of spermathecae and cibarium, after dissection of fresh samples. In this study, therefore, only female samples were used for analysis. A total of 1,480 female sand flies belonging to 25 Lutzomyia species were collected. The number of female sand flies collected was 417 (28.2%) using the mini-Shannon trap, 259 (17.5%) using the CDC light trap and 804 (54.3%) by human landing. The total number of sand flies per trap collected by the different methods was markedly affected by the study site, probably because of the various composition of species at each locality. Furthermore, as an additional study, the attraction of sand flies to mini-Shannon traps powered with LED white-light and LED black-light was investigated preliminarily, together with the CDC light trap and human landing. As a result, a total of 426 sand flies of nine Lutzomyia species, including seven man-biting and two non-biting species, were collected during three capture trials in May and June 2014 in an area endemic for leishmaniasis (La Ventura). The black-light proved relatively superior to the white-light with regard to capture numbers, but no significant statistical difference was observed between the two traps.