Identification Of Blood Meal Sources Research Articles

Molecular identification of blood meal sources in black flies (Diptera: Simuliidae) suspected as leucocytozoon vectors

Identification of the host blood meal of hematophagous insects can provide significant information regarding host preferences and the possibility of the transmission of disease agents. Currently, this knowledge is limited for black flies in the Oriental region. In this study, we used cytochrome b gene sequences for identification of blood meal sources of two black fly taxa, the Simulium asakoae complex and S. chumpornense Takaoka & Kuvangkadilok in Thailand. A total of 4,260 wild adult females were visually screened revealing 24 blood-engorged females from which cytochrome b was successfully amplified in 19 individuals. Comparisons in GenBank database revealed that all are closest to chicken (Gallus gallus) with sequence similarity of >98%. Therefore, these black fly species are feeding on chickens. We also molecularly investigated the hemosporidian blood protozoa genus Leucocytozoon in black flies and found 13 of 19 blood-engorged females positive for this protozoon. Sequence analysis revealed that this Leucocytozoon DNA could be assigned into two previously recognized groups, one with a Leucocytozoon reported from domestic chickens and black flies in Thailand, and another close to the L. schoutedeni. Our results indicate a high possibility that the S. asakoae complex and S. chumpornense are natural vectors of Leucocytozoon.

Identification of bloodmeal sources of triatomines captured in the Paraguayan Chaco region of South America by means of molecular biology analysis

ABSTRACTThe Paraguayan Chaco is an isolated environment with its own unique ecosystem. In this region, Chagas disease remains a health problem. Chagas disease is caused by the parasite Trypanosoma cruzi, and it is primarily transmitted by triatomines. In order to identify the blood meal sources of triatomines, specimens of the vector were collected in domestic and peridomestic areas and the PCR-RFLP method was implemented. Cytochrome b was amplified from the samples and later subjected to digestion with two restriction enzymes: Hae III and Xho I.It was possible to generate distinct restriction patterns on the amplified material to identify several blood meal sources for the vectors. We employed the blood from several species as positive controls: human, chicken, canine, feline, and armadillo blood. However, we identified only 3 sources for the blood meals of the insect vectors: human, chicken and canine blood. In total, 76 triatomines were captured. T. cruzi was not found in any of them. In 61% of the captured specimens, the blood meal sources for the vectors could be identified. In 30% of these cases, the presence of DNA from more than one vertebrate was detected in the same triatomine. The most common blood meal source found was chicken blood. The presence of human and chicken blood in triatomines captured in domestic and peridomestic areas strongly suggests that the parasite can freely move amongst both areas regardless of food availability. Free vector movement in these areas constitutes an epidemiological threat for the inhabitants of the community under study.

Comparison of phenol-chloroform and a commercial deoxyribonucleic acid extraction kit for identification of bloodmeal sources from triatomines (Hemiptera: Reduviidae).

INTRODUCTION: Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles. Expensive commercial deoxyribonucleic acid (DNA) extraction kits are widely used for bloodmeal identification. This study assessed the performance of an inexpensive phenol-chloroform DNA extraction protocol for identification of triatomine bloodmeal sources, comparing it with a commercially available kit. METHODS: Both methods were used to obtain DNA from the intestinal contents of Triatoma brasiliensis blood-fed on either Columba sp., Mus musculus, or Gallus gallus. Subsequently, the mitochondrial 12S ribosomal ribonucleic acid (rRNA) gene was amplified by polymerase chain reaction, sequenced, and compared with GenBank data. RESULTS: Twelve (80%) samples extracted with the commercial kit and four (26.7%) with phenol-chloroform were pure (according to the A260/A280 ratio). Samples extracted with phenol-chloroform, except for Columba sp. samples, had higher DNA concentration than those extracted with the commercial kit. Samples extracted using phenol-chloroform and blood-fed on G. gallus had significantly higher DNA concentration than those blood-fed on Columba sp. (p-value <0.001) and M. musculus (p-value <0.001). The 215-base-pair 12S rRNA fragment was amplified from all samples and produced reliable sequences, enabling the identification of the bloodmeal source, most of which showed identity and coverage above 95%. The phenol-chloroform method was much less expensive than the commercial kit but took considerably more time to perform. CONCLUSIONS: Our data showed that both DNA extraction methods produced reliable sequences enabling identification of triatomine bloodmeal sources but differed greatly in cost and time required.

An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil.

BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved.OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.

Disentangling Trypanosoma cruzi transmission cycle dynamics through the identification of blood meal sources of natural populations of Triatoma dimidiata in Yucat\xe1n, Mexico

BackgroundIn the Yucatán Peninsula, Mexico, Triatoma dimidiata is the main vector of Trypanosoma cruzi, the causative agent of Chagas disease. Little effort has been made to identify blood meal sources of T. dimidiata in natural conditions in this region, although this provides key information to disentangle T. cruzi transmission cycles and dynamics and guide the development of more effective control strategies. We identified the blood meals of a large sample of T. dimidiata bugs collected in different ecotopes simultaneously with the assessment of bug infection with T. cruzi, to disentangle the dynamics of T. cruzi transmission in the region.MethodsA sample of 248 T. dimidiata bugs collected in three rural villages and in the sylvatic habitat surrounding these villages was used. DNA from each bug midgut was extracted and bug infection with T. cruzi was assessed by PCR. For blood meal identification, we used a molecular assay based on cloning and sequencing following PCR amplification with vertebrate universal primers, and allowing the detection of multiple blood meals in a single bug.ResultsOverall, 28.7% of the bugs were infected with T. cruzi, with no statistical difference between bugs from the villages or from sylvatic ecotopes. Sixteen vertebrate species including domestic, synanthropic and sylvatic animals, were identified as blood meal sources for T. dimidiata. Human, dog and cow were the three main species identified, in bugs collected in the villages as well as in sylvatic ecotopes. Importantly, dog was highlighted as the main blood meal source after human. Dog was also the most frequently identified animal together with human within single bugs, and tended to be associated with the infection of the bugs.ConclusionsDog, human and cow were identified as the main mammals involved in the connection of sylvatic and domestic transmission cycles in the Yucatán Peninsula, Mexico. Dog appeared as the most important animal in the transmission pathway of T. cruzi to humans, but other domestic and synanthropic animals, which most were previously reported as important hosts of T. cruzi in the region, were evidenced and should be taken into account as part of integrated control strategies aimed at disrupting parasite transmission.

Association of Phlebotomus guggisbergi with Leishmania major and Leishmania tropica in a complex transmission setting for cutaneous leishmaniasis in Gilgil, Nakuru county, Kenya.

BackgroundPhlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area.Methodology/ Principal findingsAn entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider’s insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans.Conclusions/ SignificanceThe high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area.

Molecular identification of bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp. trapped on cattle farm settlements in southwest Nigeria.

The interactions of host, vector and parasite in bovine trypanosomiasis transmission cycles in southwest Nigeria are not yet well understood. Trypanosoma (Trypanosomatida: Trypanosomatidae) species infection prevalences and bloodmeal sources were determined in transmitting vectors of the genera Glossina (Diptera: Glossinidae), Tabanus (Diptera: Tabanidae) and Stomoxys (Diptera: Muscidae) collected using Nzi traps in cattle settlements in southwest Nigeria. Sequenced cytochrome B mitochondrial DNA segments obtained from vector digestive tracts identified bloodmeal sources from eight host species, namely human, cattle, hippopotamus, giraffe, gazelle, spotted hyena, long-tailed rat and one unidentified species. Overall, 71.1% [95% confidence interval (CI) 63.0-78.1], 33.3% (95% CI 21.9-47.0) and 22.2% (95% CI 16.2-29.9), respectively, of Glossina, Tabanus and Stomoxys flies were positive for trypanosomes. The observed trypanosome species were Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma evansi, Trypanosoma simiae and Trypanosoma godfreyi. Trypanosome DNA was more prevalent in tsetse (34.8% Tr.vivax, 51.1% Tr.b. brucei, 5.2% Tr.congolense, 4.4% Tr.simiae and 24.4% mixed infections) than in other flies and the main determinants in all flies were seasonal factors and host availability. To the best of the present group's knowledge, this is the first report of Trypanosoma species in Tabanus and Stomoxys flies in Nigeria. It indicates that vector control programmes should always consider biting flies along with tsetse flies in the control of human and animal trypanosomiasis.

Genetic diversity of trypanosomes pathogenic to livestock in tsetse flies from the Nech Sar National Park in Ethiopia: A concern for tsetse suppressed area in Southern Rift Valley?

In Ethiopia, home to the largest African herd of cattle, animal trypanosomiasis is a major constraint to the efforts made for food self-sufficiency. We searched for trypanosomes in tsetse flies caught in the Nech Sar National Park (NSNP), Southern Rifty Valley, Ethiopia, at the district of Arba Minch where intensive tsetse control is successfully improving cattle productivity. Despite narrow geographical and temporal scales of our survey, we found a remarkable diversity of trypanosomes using the sensitive and discriminative method of fluorescent fragment length barcoding. We also found a high density of Glossina pallidipes (47.8 flies/trap/day) showing relevant cytochrome oxidase I gene variability. The identification of blood meal sources through cytochrome b gene sequences revealed cattle and warthog as preferential ungulate hosts of tsetse flies in the study area. Our survey identified trypanosomes in 38% of the 287 flies examined (42% of proboscises and 32% of guts), and the following infection rates for each species: Trypanosoma vivax 23%, T. simiae 23%, T. congolense 22%, T. theileri 19.9%, T. (Trypanozoon) spp. 10.5%, T. godfreyi 9.4%, T. simiae Tsavo 6.3%, and mixed infections in proboscises (30%) and guts (61%). Phylogenetic analysis revealed T. vivax of the “West African-South American” genotype, T. congolense of Savannah (16.7%), Kilifi (3.5%) and Forest (2.1%) lineages, and new genotypes of T. simiae. To our knowledge, this is the first survey of trypanosomes in the NSNP, and the most comprehensive molecular characterisation of trypanosomes in tsetse flies of Ethiopia, including the comparison with samples from West and other East African countries. Our results support the diversification of T. vivax in East Africa, and the dispersion of the genotype herein identified in Ethiopia across West Africa and then in South America. Altogether, tsetse density and infection rate, repertoire of trypanosomes and feeding behavior indicate a high risk of transmission of trypanosomes pathogenic to ungulates by tsetse flies from the NSNP, a hotspot of tsetse infestation and trypanosome diversity. Our findings reinforce the need for constant surveillance, and the reliance on community efforts to prevent reinvasion of tsetse and animal trypanosomiasis in suppressed areas of Southern Rift Valley.

Molecular bloodmeal analyses reveal that Trypanosoma cruzi-infected, native triatomine bugs often feed on humans in houses in central Brazil.

The identification of bloodmeal sources in triatomine bugs (Hemiptera: Reduviidae) is important in understanding vector-host associations and in measuring the risk for Chagas' disease transmission. The bloodmeal sources of triatomines infected with Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae) caught in houses in central Brazil (Goiás State and the Federal District) were investigated during 2012-2014. Mitochondrial cytochrome b amplicons were used to identify bloodmeals through high-resolution melting and DNA sequencing. Most bugs were found to have fed on either humans (45.7%) or chickens (43.1%). Human blood was detected in Triatoma sordida (n = 22/50 bugs), Triatoma pseudomaculata (n = 7/11 bugs), Panstrongylus megistus (n = 10/24 bugs), Panstrongylus geniculatus (n = 1/3 bugs) and Rhodnius neglectus (n = 18/28 bugs) (all: Hemiptera: Reduviidae). Sequencing identified Necromys (Rodentia: Cricetidae) mouse blood in P. geniculatus and Tropidurus (Squamata: Tropiduridae) lizard blood in T. pseudomaculata and T. sordida. These findings reveal new vector-host associations. The present results suggest frequent contact between humans and T. cruzi-infected triatomines in central Brazil and indicate that Chagas' disease transmission by native vectors is an ongoing threat.

Identification of Blood Meals from Potential Arbovirus Mosquito Vectors in the Peruvian Amazon Basin.

The transmission dynamics of many arboviruses in the Amazon Basin region have not been fully elucidated, including the vectors and natural reservoir hosts. Identification of blood meal sources in field-caught mosquitoes could yield information for identifying potential arbovirus vertebrate hosts. We identified blood meal sources in 131 mosquitoes collected from areas endemic for arboviruses in the Peruvian Department of Loreto by sequencing polymerase chain reaction amplicons of the cytochrome b gene. Psorophora (Janthinosoma) albigenu, Psorophora (Grabhamia) cingulata, Mansonia humeralis, Anopheles oswaldoi s.l., and Anopheles benarrochi s.l. had mainly anthropophilic feeding preferences; Aedes (Ochlerotatus) serratus, and Aedes (Ochlerotatus) fulvus had feeding preferences for peridomestic animals; and Culex (Melanoconion) spp. fed on a variety of vertebrates, mainly rodents (spiny rats), birds, and amphibians. On the basis of these feeding preferences, many mosquitoes could be considered as potential enzootic and bridge arbovirus vectors in the Amazon Basin of Peru.

Characterization of an Indoor-Resting Population ofAnopheles arabiensis(Diptera: Culicidae) and the Implications on Malaria Transmission in Tubu Village in Okavango Subdistrict, Botswana

Comprehensive knowledge on vector dynamics is lacking in Botswana and yet essential for effective indoor residual spraying. This study assessed some of the entomological indices that contribute to malaria transmission by an indoor-resting population of Anopheles arabiensis Patton (Diptera: Culicidae) in Tubu village, Okavango subdistrict. The pyrethroid space-spray technique and hut searches were used to sample mosquitoes. Species and bloodmeal source identification were done using the polymerase chain reaction techniques. The infective status was determined by the enzyme-linked immuno-sorbent assay test. The human blood indices (HBI), human-biting rates (HBR), and vector densities were computed. Anopheles arabiensis was the sole vector and member of the Anopheles gambiae Giles complex identified. Significant changes in vector densities were observed over seasons, while nonsignificant differences were observed among the huts (P > 0.05). The main source of bloodmeal was cattle (46.8% [65]). There were no significant differences in HBI (P > 0.05) and HBR (P > 0.05) between the seasons. All the 271 mosquitoes tested for sporozoite infection were negative. The results showed that seasonal variations in vector densities corresponded to the traditional malaria and nonmalaria seasons. The vector population was relatively more zoophagic. The HBI and HBR were not influenced by the seasons. None of the endophilic mosquitoes tested (N = 271) was positive for sporozoites. Our study provided some relevant basic information to the local malaria control program that may be used to strategize their operations if malaria elimination has to be achieved by 2017.

Identification of blood meal sources in the main African malaria mosquito vector by MALDI-TOF MS.

BackgroundThe identification of blood meal sources in malaria vectors is critical to better understanding host/vector interactions and malaria epidemiology and control. Currently, the identification of mosquito blood meal origins is based on time-consuming and costly techniques such as precipitin tests, ELISA and molecular tools. Although these tools have been validated to identify the blood meal and trophic preferences of female Anopheles mosquitoes, they present several limitations. Recently, matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successfully used as a quick and accurate tool for arthropod identification, including mosquitoes. The aim of the present work was to test whether MALDI-TOF MS could also be applied to identification of blood meal sources from engorged mosquitoes.MethodsAbdomen proteins extracted from Anopheles gambiae (stricto sensu, S molecular form) that were either unengorged or artificially engorged on seven distinct types of vertebrate blood (human, horse, sheep, rabbit, mouse, rat, dog) were submitted for MALDI-TOF MS.ResultsThe comparison of mass spectrometry (MS) spectra from mosquito abdomens collected 1 h post-feeding, were able to discriminate blood meal origins. Moreover, using Aedes albopictus specimens, abdominal protein MS spectra from engorged mosquitoes were found specific to host blood source and independent of the mosquito species. A sequential analysis revealed stability of mosquito abdominal protein spectra up to 24 h post-feeding.ConclusionsThese results indicate that MALDI-TOF MS could determine feeding patterns of freshly engorged mosquitoes up to 24 h post-blood meal. The MALDI-TOF MS technique appears to be an efficient tool for large epidemiological surveillance of vector-borne diseases and outbreak source identification.

Foraging range of arthropods with veterinary interest: New insights for Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae) using the ring method

The identification of blood meal source of arthropod vector species contributes to the understanding of host-vector-pathogen interactions. The aim of the current work was to identify blood meal source in Culicoides biting midge species, biological vectors of internationally important arboviruses of livestock and equids, using a new ecological approach. We examined the correlation between blood meal source identified in engorged Culicoides females collected in a suction light trap and the available vertebrate hosts along four rings (200, 500, 1000 and 2000m) centered at the trap site and described the foraging range of the three main vector species of veterinary interest present in the study area, Culicoides imicola, Culicoides kingi and Culicoides oxystoma. The study was performed in four sites localized in the Niayes region of Senegal (West Africa) where recent outbreaks of African horse sickness occurred. Blood meal source identification was carried out by species-specific multiplex PCRs with genomic DNA extracted from the abdomen of engorged females collected during nine night collections for twenty-six collections. The four most abundant hosts present in the studied area (horse, cattle, goat and sheep) were surveyed in each ring zone. The blood meal source varied according to Culicoides species and host availability in each site. C. oxystoma and C. imicola females mainly fed on horses readily available at 200m maximum from the trap location whereas females of C. kingi fed mainly on cattle, at variable distances from the traps (200 to 2000m). C. oxystoma may also feed on other vertebrates. We discuss the results in relation with the transmission of Culicoides-borne arboviruses and the species dispersion capacities.

English

There is a steady increase in the contact between humans and wildlife, brought about by encroachment, destruction of natural forests, climatic and environmental changes. Mosquitoes get exposed to hosts and pathogens; creating possibilities for new disease patterns. Therefore, the identification of blood-meal sources is important to determine the linkages between hosts and vectors. Engorged mosquitoes were collected in Zika forest (Uganda) for a period of 12 months using carbon dioxide (CO2)-baited light traps. Total genomic DNA was extracted from the abdominal contents of the mosquitoes and the diagnostic regions of the mitochondrial genes, cytochrome oxidase subunit 1 (COI) and cytochrome b (cytb) sequenced. The sequences were subsequently blasted in the GenBank. Sequence analyses revealed that feeds were derived from mammalian and avian hosts. Blood-meal sources from Aedes africanus (Theobold) examined were entirely human. There were significant differences between host species from which the mosquito species fed (Krusakal Wallis test, χ2 = 19.118, df = 5, p = 0.018). Several mosquitoes were considered as potential bridge vectors for a number of arboviruses and other diseases have been collected from the forest. Taking of mammalian origin blood-meals, including humans, may facilitate exposure to new pathogens and disease patterns. Key words: Blood-meal, bridge vectors, cytochrome oxidase sub unit I, cytochrome b, Entebbe.

Molecular identification of blood meal sources of ticks (Acari, Ixodidae) using cytochrome b gene as a genetic marker.

Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91–98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamys sabanus, Rattus tiomanicus and Sundamys muelleri), shrews (Tupaia glis) and mammals (Tapirus indicus and Prionailurus bengalensis), supported by 82–88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks’ blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.

Eco-epidemiology of Chagas disease in an endemic area of Colombia: risk factor estimation, Trypanosoma cruzi characterization and identification of blood-meal sources in bugs.

The Sierra Nevada de Santa Marta (SNSM) is a mountainous area in Colombia that is highly endemic to Chagas disease. We explored some eco-epidemiological attributes involved in the Chagas disease transmission scenario in three Indigenous communities. An epidemiological survey was done, where parasite infection in reservoirs and insects, Trypanosoma cruzi genotyping, identification of blood-meal sources in intradomiciliary insects using the high-resolution melting technique, and some risk factors were evaluated. The results suggest that several dwelling conditions such as thatched palm roofs and mud walls carried the highest risk of finding intradomiciliary Rhodnius prolixus, which 56.41% were infected with T. cruzi and fed with human blood. Moreover, T. cruzi Ia was the most frequent haplotype found in insects. These results indicate the existence of a domestic T. cruzi transmission cycle that does not overlap with the sylvatic cycle, and highlight the need for efficient entomological control focused to this area.

Anopheles (Kerteszia) cruzii (Diptera: Culicidae) in peridomiciliary area during asymptomatic malaria transmission in the Atlantic Forest: molecular identification of blood-meal sources indicates humans as primary intermediate hosts.

Anopheles (Kerteszia) cruzii has been implicated as the primary vector of human and simian malarias out of the Brazilian Amazon and specifically in the Atlantic Forest regions. The presence of asymptomatic human cases, parasite-positive wild monkeys and the similarity between the parasites infecting them support the discussion whether these infections can be considered as a zoonosis. Although many aspects of the biology of An. cruzii have already been addressed, studies conducted during outbreaks of malaria transmission, aiming at the analysis of blood feeding and infectivity, are missing in the Atlantic Forest. This study was conducted in the location of Palestina, Juquitiba, where annually the majority of autochthonous human cases are notified in the Atlantic Forest of the state of São Paulo. Peridomiciliary sites were selected for collection of mosquitoes in a perimeter of up to 100 m around the residences of human malaria cases. The mosquitoes were analyzed with the purpose of molecular identification of blood-meal sources and to examine the prevalence of Plasmodium. A total of 13,441 females of An. (Ker.) cruzii were collected. The minimum infection rate was calculated at 0.03% and 0.01%, respectively, for P. vivax and P. malariae and only human blood was detected in the blood-fed mosquitoes analyzed. This data reinforce the hypothesis that asymptomatic human carriers are the main source of anopheline infection in the peridomiciliary area, making the probability of zoonotic transmission less likely to happen.

Identification of blood meal sources of Lutzomyia longipalpisusing polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene.

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Hemi-Nested PCR and RFLP Methodologies for Identifying Blood Meals of the Chagas Disease Vector, Triatoma infestans

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.- Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.- restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.

Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on Palaearctic biting midge species, vectors of Orbiviruses

Culicoides are small biting midges involved worldwide in the transmission of bluetongue and African horse sickness viruses. Feeding behaviours of Palaearctic biting midge species and their spatio-temporal dynamics remain unclear at the specific level. Three multiplex species-specific PCR-based assays were developed and used to identify blood meal source of engorged females of Palaearctic midge species of veterinary interest. Species-specific primers of potential hosts from livestock, domestic animals and wildlife (cattle, goat, sheep, red deer, roe deer, chamois, dog, pig, cat, horse) were designed and multiplexed from the mitochondrial cytochrome b gene. The assays also make possible to identify whether multiple blood meals have been taken. The first results from several Culicoides populations sampled in France highlight the utility of this valuable diagnostic tool combined with species identification assays, and suggest that most of the Culicoides species may have an opportunistic feeding behaviour regarding the host distribution and density. Noteworthy is the peculiar trophic behaviour of Culicoides chiopterus showing clear trends to cattle. Information on host preference and feeding behaviours are crucial for a better understanding of vector–host interactions and disease epidemiology.