Genetic similarities and phylogenetic analysis of Muntjac (Muntiacus spp. ) by comparing the nucleotide sequence of 16S rRNA and cytochrome B genome / Semelhanças genéticas e análise filogenética de muntjac (Muntiacus spp. ) comparando a sequência de nucleótidos de rRNA 16S e genoma citocromo B

This study aimed to identify the phylogenetic similarities among the muntjac (Muntiacus spp.). The phylogenetic similarities among seven major muntjac species were studied by comparing the nucleotide sequence of 16s rRNA and cytochrome b genome. Nucleotide sequences, retrieved from NCBI databases were aligned by using DNASTAR software. A phylogenetic tree was created for the selected species of muntjac by using the maximum likelihood method on MEGA7 software. The results of nucleotide sequences (16s rRNA) showed phylogenetic similarities between, the M. truongsonensis and M. rooseveltorum had the highest (99.2%) while the lowest similarities (96.8%) found between M. crinifrons and M. putaoensi. While the results of nucleotide sequences (Cty b) showed the highest similarity (100%) between M. muntjak and M. truongsonensis and the lowest s (91.5%) among M. putaoensis and M. crinifrons. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including M. putaoensis, M. truongsonensis, M. rooseveltorum, and M. muntjak, and the second one including M. crinifrons and M. vuquangensis. The M. reevesi exists separately in the phylogenetic tree. The phylogenetic tree of muntjac species using cytochrome b genes shows that the M. muntjak and M. truongsonensis are clustered in the same group.

Este estudo visou identificar as semelhanças filogenéticas entre os muntjac (Muntiacus spp.). As semelhanças filogenéticas entre sete grandes espécies muntjac foram estudadas comparando a sequência de nucleótidos de 16s rRNA e genoma citocromo b. As sequências de nucleótidos, obtidas a partir de bases de dados NCBI, foram alinhadas utilizando o software DNASTAR. Foi criada uma árvore filogenética para as espécies selecionadas de muntjac utilizando o método de probabilidade máxima no software MEGA7. Os resultados das sequências de nucleótidos (16s rRNA) mostraram semelhanças filogenéticas entre o M. truongsonensis e o M. rooseveltorum tiveram o maior número (99,2%) enquanto as semelhanças mais baixas (96,8%) encontradas entre M. crinifrons e M. putaoensi. Enquanto os resultados das sequências de nucleótidos (Cty-b) apresentaram a maior semelhança (100%) entre M. muntjak e M. truongsonensis e os mais baixos (91,5%) entre M. putaoensis e M. crinifrons. A árvore filogenética das espécies muntjac (gene rRNA 16s) mostra os dois principais aglomerados, o que inclui M. putaoensis, M. truongsonensis, M. rooseveltorum e M. muntjak, e o segundo incluindo M. crinifrons e M. vuquangensis. O M. reevesi existe separadamente na árvore filogenética. A árvore filogenética das espécies muntjac usando genes citocromo b mostra que os M. muntjak e M. truongsonensis estão agrupados no mesmo grupo.

Redescription of Paradiplozoon opsariichthydis (Jiang, Wu et Wang 1984) Jiang, Wu et Wang, 1989 (Monogenea, Diplozoidae).

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis.

New morphological and molecular data for Haemoproteus (H.) paramultipigmentatus in the Atlantic Forest of Brazil.

Parasites of the genus Haemoproteus have been reported in almost all avian clades and zoogeographic regions, except Antarctica. However, despite the large number of reports worldwide, they are poorly studied in the Neotropical region, which includes the Atlantic Forest, a biodiversity hotspot with more than 890 bird species, of which 24% are endemic. Haemoproteus (Haemoproteus) paramultipigmentatus was described by morphological and molecular analyses in 2013 infecting Columbiform birds in Mexico. However, since the original description this parasite has not been studied in detail. Here, we investigate the prevalence of Haemoproteus spp. in Brazilian Columbiformes and conducted a taxonomic integrative study of the species Haemoproteus (Haemoproteus) paramultipigmentatus, including new morphological and molecular data from a Brazilian population. Moreover, we provide discussions about the geographic distribution and phylogenetic relationships between different lineages of this parasite. Our findings demonstrated a high prevalence of Haemoproteus spp. infection in Brazilian Columbiformes, which is in accordance with previous studies. Morphological characterization of H. paramultipigmentatus revealed minor differences from the original description. Through molecular and phylogenetic analyses we identified a new lineage of H. paramultipigmentatus that was added to the genetic databases. Our findings also suggest a new geographical distribution for this hemoparasite, including South American countries, and raise discussions about its current distribution.

Detection and molecular characterization of Babesia sp. in wild boar (Sus scrofa) from western Japan.

Wild animals often act as reservoirs of tick-borne Babesia and Theileria spp., which cause piroplasmosis. Therefore, epidemiological investigations about the distribution of these parasites in wild animals are important for evaluating the transmission risk to humans and livestock. In this study, we surveyed Babesia and Theileria spp. infecting wild boar (Sus scrofa) in Kagoshima and Yamaguchi prefectures and Tsushima island, which are all in western Japan, and performed molecular genetic analyses on the samples. DNA was extracted from either blood or liver samples of wild boar captured in Kagoshima prefecture in 2015, 2016, and 2018 and from blood samples from wild boar captured in Yamaguchi prefecture in 2013-2015 and Tsushima island in 2018. PCR screening for the partial 18S ribosomal RNA gene (18S rRNA) of both Babesia and Theileria spp. in wild boar revealed that 63.9 % (140 of 219 samples) were positive. Sequencing of all positive samples revealed that they were all the same Babesia species. Subsequent phylogenetic analyses showed that the parasite is closely related to Babesia sp. previously detected in the hard tick, Amblyomma testudinarium in Kagoshima, and further analyses suggested that this species is genetically related to Babesia gibsoni. On the other hand, no Theileria were detected in any of the samples. In summary, we observed a high prevalence of B. gibsoni-like Babesia sp. in wild boar in western regions of Japan. The host range, distribution, pathogenicity, and life cycle of this protozoan should be further evaluated.

Cytochrome b as a more promising marker for analysing the distribution vector for Metagonimus suifunensis (Trematoda: Heterophyidae).

In this study of Metagonimus suifunensis (M. suifunensis) in the Russian Southern Far East, the variability of the full-length sequences of the cytochrome b (cytb) mtDNA gene was assessed for the first time. In addition, the cox1 mtDNA gene sequences were also obtained for this species from new localities. In total, 87 and 81 sequences of the cytb and cox1 genes, respectively, were used in the current study. The cytb gene proved more promising and revealed two haplogroups that are associated with the spatial distribution of the species: geographical isolation caused the fixation of differences between northern and southern populations. In addition, the results obtained for the cytb gene opened up new perspectives in the analysis of sequences of the cox1 gene, which was not sufficiently effective as a sole marker. Based on data for both mitochondrial genes, molecular processes influencing the formation of the modern population were analysed for M. suifunensis. The new data confirmed the previously expressed opinion that this species colonized the study territory from north to south and will form the basis for determining possible ways of its further expansion, which is important for predicting the emergence of new foci of metagonimosis.

Chromosomal Diversity in Two Allopatric Populations of Farlowella hahni Meinken 1937 (Teleostei: Siluriformes): Cytogenetics and Cytochrome b Analyses.

Farlowella is the second richest genus in Loricariinae, broadly distributed in freshwater streams and rivers of South America. In this article, we aimed to expand on the cytogenetic and molecular data available for two allopatric populations of Farlowella hahni. Both populations had diploid chromosome number 58, but with karyotype differences, indicative of chromosomal rearrangements. C-banding showed large heterochromatic blocks at telomeric regions in acrocentric chromosomes in both populations. Fluorescence in situ hybridization (FISH) revealed a single 18S rDNA site in both populations and a single 5S rDNA site for individuals from lower Paraná River basin (native region) and multiple 5S rDNA sites for individuals from upper Paraná River basin (non-native region). Mitochondrial sequence analyses did not separate the two F. hahni populations. The cytogenetic and molecular data obtained are relevant in a preliminary study and suggested the existence of cryptic diversity and the hypothesis that at least two Farlowella lineages may coexist in the Paraná basin.

An unpleasant souvenir: Endoscopic finding of Trichuris trichiura (Nematoda: Trichuridae).

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.

A new species of Triplophysa (Cypriniformes, Nemacheilidae) from Weihe River in Gansu Province, China.

A new species of Tibetan loach, Triplophysa weiheensis sp. nov., is described from the Weihe River in Gansu Province, China, based on morphological and molecular analyses. The new species can be distinguished from all known congeners by a unique combination of the following characters: scaleless; snout abruptly sloping downward, anterior to anterior nostril; lower jaw crescentic, not sharp; body without obvious mottling; lateral line interrupted on posterior trunk at pelvic-fin distal extremity; caudal-peduncle length 2.0-2.7 times its depth; branched rays of pectoral fin 10-11; branched rays of pelvic fin 5-6; inner gill rakers on 1 st gill arch 14-16; vertebrae 4+34-36; intestine with 6-7 loops, length ca. 1.8 times SL ( n=3); bony capsule of air bladder small and thin; posterior chamber of air bladder absent.

A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene.

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/µL). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs.

Molecular characterization of Trichuris spp. from captive animals based on mitochondrial markers.

Monoxenous parasites may easily infect animals in captivity, and nematodes belonging to the genus Trichuris are commonly reported in zoological gardens worldwide. Infections in captive animals should be accurately monitored and the characterization of pathogens is highly advisable, as a tool to infer possible routes of intra- and interspecific transmission pathways and to assess the related zoonotic potential. Whipworms are usually identified on the basis of few morphological features of adults males and eggs and by an host-affiliation criterion. Given the strong morphological convergence of adaptive traits and the possible occurrence of hybridization and/or cross-infections events, the use of molecular methods is of great utility. Here, we analysed two partial mitochondrial loci, the cytochrome c oxidase I and the cytochrome b regions, in Trichuris spp. infecting four animal species hosted in the Bioparco Zoological Garden of Rome. Results from molecular systematics, compared to previous data, suggested that the five Trichuris taxa recovered were well separated, showing a significant degree of host affiliation (herbivorous, primates/swine and rodents/canids). The screw horn antelopes and the camels were infected with two variants of Trichuris ovis; Trichuris sp. similar to those observed in rodents from South America was infecting the Patagonian maras. Moreover, Trichuris from the ring-tailed lemur showed a great similarity to Trichuris infecting the Japanese macaque previously analysed from the same zoological garden, and clustering together with Trichuris trichiura, posing a potential zoonotic threat for visitors and workers.

First report of malaria parasites in water buffalo in Nepal.

We present the first molecular-based report on ungulate malaria parasites from water buffalo in Nepal. Fifty-six blood samples were collected from different groups of water buffalo (wild, feral, and domestic) and PCR assays were conducted using Plasmodium spp. cytb specific primers. Two positive cases were detected, one each from feral and domestic individuals. Complete mitochondrial genome sequence (5987 bp) was obtained and examined for nucleotide variations. Sequence analysis revealed identity with type II water buffalo malaria parasites, reported previously, with one A to T nucleotide difference at position 5344. Prevalence, as well as possible economic impacts of water buffalo malaria, should be determined on a wider set of samples from buffalo across Nepal.

Species Boundaries within Morphologically Cryptic Galagos: Evidence from Acoustic and Genetic Data.

Describing primate biodiversity is one of the main goals in primatology. Species are the fundamental unit of study in phylogeny, behaviour, ecology and conservation. Identifying species boundaries is particularly challenging for nocturnal taxa where only subtle morphological variation is present. Traditionally, vocal signals have been used to identify species within nocturnal primates: species-specific signals often play a critical role in mate recognition, and they can restrict gene flow with other species. However, little research has been conducted to test whether different "acoustic forms" also represent genetically distinct species. Here, we investigate species boundaries between two putative highly cryptic species of Eastern dwarf galagos (Paragalago cocosand P. zanzibaricus). We combined vocal and genetic data: molecular data included the complete mitochondrial cytochrome b gene (1,140 bp) for 50 samples across 11 localities in Kenya and Tanzania, while vocal data comprised 221 vocalisations recorded across 8 localities. Acoustic analyses showed a high level of correct assignation to the putative species (approx. 90%), while genetic analyses identified two separate clades at the mitochondrial level. We conclude that P. cocos and P. zanzibaricus represent two valid cryptic species that probably underwent speciation in the Late Pliocene while fragmented in isolated populations in the eastern forests.

On the true identity of Sergentomyia gemmea and description of a closely related species: Se. raynali n. sp.

Several species of Leishmania are responsible for leishmaniases in Thailand, although little is known about their transmission. Sergentomyia gemmea has been suspected several times to transmit Leishmania martiniquensis. Some captures carried out in Thailand and Lao People's Democratic Republic have emphasized the scarcity of Se. gemmea, comprising only 1% of the collected females. The sequencing of cytochrome B mtDNA of our specimens showed that our specimens are not grouped with other Se. gemmea previously deposited in GenBank. The latter are grouped with some Se. khawi and Se. hivernus that we processed in the present study. We suspect misidentifications and propose focusing on the most useful characters for identification of Se. gemmea based on the examination of type-specimens. The examination of the ascoids exhibiting anterior spurs is the most important one. However, we also describe Se. raynali n. sp. exhibiting comparable spurs but differing from Se. gemmea by its original cibarium. Finally, the vectorial role of Se. gemmea appears very questionable in the absence of new evidence.

High susceptibility of the laboratory-reared biting midges Culicoides nubeculosus to Haemoproteus infections, with review on Culicoides species that transmit avian haemoproteids.

Haemosporidian parasites belonging to Haemoproteus cause avian diseases, however, vectors remain unidentified for the majority of described species. We used the laboratory-reared biting midges Culicoides nubeculosus to determine if the sporogonic development of three widespread Haemoproteus parasites completes in this insect. The midges were reared and fed on one common blackbird, white wagtail and thrush nightingale naturally infected with Haemoproteus minutus, Haemoproteus motacillae and Haemoproteus attenuatus, respectively. The engorged females were dissected in order to follow their sporogonic development. Microscopic examination was used to identify sporogonic stages. Bayesian phylogeny based on partial cytochrome b gene was constructed in order to determine phylogenetic relationships among Culicoides species-transmitted haemoproteids. All three parasites completed sporogony. Phylogenetic analysis placed Culicoides species transmitted haemoproteids in one well-supported clade, proving that such analysis readily indicates groups of dipteran insects transmitting avian haemoproteids. Available data show that 11 species of Culicoides have been proved to support complete sporogony of 18 species of avian haemoproteids. The majority of Culicoides species can act as vectors for many Haemoproteus parasites, indicating the low specificity of these parasites to biting midges, whose are globally distributed. This calls for control of haemoproteid infections during geographical translocation of infected birds.

Polychromophilus spp. (Haemosporida) in Malagasy bats: host specificity and insights on invertebrate vectors.

BACKGROUND: Bats are home to diverse haemosporidian parasites namely Plasmodium and Plasmodium-related. While information is available at a worldwide level, haemosporidian infection in bats from Madagascar is still scarce and recent changes in the taxonomy of the island's bat fauna, particularly the description of several new species, require a reassessment of previously described patterns, including blood parasite ecology and vectorial transmission. METHODS: A sample representing seven of the nine known bat families and 31 of the 46 currently recognized taxa from Madagascar and collected in the western and central portions of the island were screened by PCR for the presence of Polychromophilus. In addition, Nycteribiidae flies parasitizing Miniopteridae and Vespertilionidae were screened for parasites with the aim to better understand aspects of vector transmission. Phylogenetic reconstruction using the mitochondrial cytochrome b encoding gene was used in a Bayesian analysis to examine the relationship between Polychromophilus recovered from Malagasy bats and those identified elsewhere. RESULTS: Polychromophilus infection was restricted to Miniopterus spp. (Miniopteridae), Myotis goudoti (Vespertilionidae), and Paratriaenops furculus (Rhinonycteridae), with an overall infection rate of 13.5%. Polychromophilus melanipherus was found infecting Miniopterus spp. and P. furculus, whereas Polychromophilus murinus was only recovered from M. goudoti. These two protozoan parasites species were also detected in bat flies species known to parasitize Miniopterus spp. and M. goudoti, respectively. Generalized linear model analyses were conducted to elucidate the effect of species and sex on haemoparasites infection in Miniopterus spp., which revealed that males have higher risk of infection than females and prevalence differed according to the considered Miniopterus host. Molecular screening of nycteribiid flies revealed three positive species for Polychromophilus spp., including Penicillidia sp. (cf. fulvida), Penicillidia leptothrinax, and Nycteribia stylidiopsis. These three fly species are known to parasitize Miniopterus spp. and M. goudoti and should be considered as potential vectors of Polychromophilus spp. CONCLUSION: Phylogenetic analyses demonstrated the existence of at least four distinct clades within the genus Polychromophilus, two of which were documented in the present study. The screening of nycteribiid flies overlaid on the highly diversified genus Miniopterus, provides considerable insight into parasite transmission, with bat infection being associated with their roosting behaviour and the occurrence of specific arthropod vectors.

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria.

BACKGROUND: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. METHODS: The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. RESULTS: Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. CONCLUSION: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

The Taxonomic Status of Spermophilus in the Plague Area of Dingbian County, Shaanxi Province, China.

This study was conducted to define the taxonomic status of Spermophilus in the plague area of Dingbian County in Shaanxi Province, China, through the two-factor variance analysis of morphological characteristics, DNA barcoding, and chromosome karyotype analysis. The Spermophilus samples collected from Dingbian and Zhengxiang Baiqi Counties exhibited significant differences in their morphological measurements. All Spermophilus samples form two distinct branches in neighbor-joining (NJ) tree. One branch included the Spermophilus samples collected from Inner Mongolia, and the other branch included samples collected from the plague foci of Shaanxi Province and the Ningxia Region. The Spermophilus samples collected from Dingbian County had a chromosome number of 2n = 38 in 84.40% of all their cells. The Spermophilus species collected from the plague area of Dingbian County was categorized as Spermophilus alashanicus (S.alashamicus). The findings reported in this study are epidemiologically significant for monitoring plague in this region of west-central China.

Blood parasite infections in a wild population of ravens (Corvus corax) in Bulgaria.

BACKGROUND: Blood parasites have been studied intensely in many families of avian hosts, but corvids, a particularly cosmopolitan family, remain underexplored. Haemosporidian parasites of the common raven (Corvus corax) have not been studied, although it is the largest, most adaptable, and widespread corvid. Genetic sequence data from parasites of ravens can enhance the understanding of speciation patterns and specificity of haemosporidian parasites in corvids, and shed light how these hosts cope with parasite pressure. METHODS: A baited cage trap was used to catch 86 ravens and a nested PCR protocol was used to amplify a 479 bp fragment of the haemosporidian cytochrome b gene from the samples. The obtained sequences were compared with the MalAvi database of all published haemosporidian lineages and a phylogenetic tree including all detected raven parasites was constructed. An examination of blood smears was performed for assessment of infection intensity. RESULTS: Twenty blood parasite lineages were recovered from ravens caught in a wild population in Bulgaria. The prevalence of generalist Plasmodium lineages was 49%, and the prevalence of Leucocytozoon lineages was 31%. Out of 13 detected Leucocytozoon lineages six were known from different corvids, while seven others seem to be specific to ravens. A phylogenetic reconstruction suggests that Leucocytozoon lineages of ravens and other corvids are not monophyletic, with some groups appearing closely related to parasites of other host families. CONCLUSIONS: Several different, morphologically cryptic groups of Leucocytozoon parasites appear to infect corvids. Ravens harbour both generalist corvid Leucocytozoon as well as apparently species-specific lineages. The extraordinary breeding ecology and scavenging lifestyle possibly allow ravens to evade vectors and have relatively low blood parasite prevalence compared to other corvids.

[Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi].

INTRODUCTION: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. OBJECTIVE: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. MATERIALS AND METHODS: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. RESULTS: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. CONCLUSION: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

Estimación del tiempo límite de detección del gen citocromo b de humanos en hembras de Lutzomyia evansi / Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi

Resumen Introducción. Las técnicas de biología molecular han permitido ampliar el conocimiento sobre las fuentes de ingestión de sangre de los insectos vectores. Sin embargo, la utilidad de estas técnicas depende de la cantidad de sangre ingerida y del proceso de digestión en el insecto. Objetivo. Determinar el tiempo límite de detección del gen citocromo b (Cyt b) de humanos en hembras de Lutzomyia evansi alimentadas experimentalmente. Materiales y métodos. Se evaluaron ocho grupos de hembras de L. evansi alimentadas con sangre humana, las cuales fueron sacrificadas en intervalos de 24 horas desde el momento de la ingestión sanguínea. Se extrajo el ADN total de cada hembra y se amplificó un segmento de 358 pb del gen Cyt b. Los productos amplificados fueron sometidos a un análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP), con el fin de descartar falsos positivos. Resultados. El segmento del gen Cyt b de humanos fue detectado en 86 % (49/57) de las hembras de L. evansi a partir de las 0 horas y hasta 168 horas después de la ingestión de sangre. En 7 % (4/57) de los individuos se amplificó el ADN del insecto y en el 7 % restante no se amplificó la banda de interés. No se encontraron diferencias estadísticas en cuanto a la amplificación del segmento del gen Cyt b de humanos ni al número de muestras amplificadas entre los grupos de hembras sacrificadas a distintas horas después de la ingestión. Conclusión. El segmento del gen Cyt b de humanos fue detectable en hembras de L. evansi hasta 168 horas después de la ingestión de sangre.

Abstract Introduction: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. Objective: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Materials and methods: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. Results: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. Conclusion: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.