genetic-manipulation-ku80-strains-for-functional-genomic-analysis

Genetic Manipulation in <em>&Delta;ku80</em> Strains for Functional Genomic Analysis of <em>Toxoplasma gondii</em>

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

The Geisel School of Medicine at Dartmouth

Genetic variability in the Trypanosoma cruzi I group has recently been revealed in Colombian isolates from humans, reservoirs and vectors. Genomic rearrangements and the polymorphic regions in taxonomic markers, such as the miniexon gene, have led to the development of molecular tools to identify phylogenetic haplotypes in T. cruzi isolates. From genetic polymorphisms found in T. cruzi I isolates, they have been classified into four haplotypes according to their epidemiologic transmission cycles. Haplotype Ia is associated with domestic isolates, from Rhodnius prolixus; haplotype Ib, with the domestic and peridomestic cycle, mainly associated with Triatoma dimidiata; haplotype Ic is a poorly characterized group, which has been associated with the peridomestic cycle; and haplotype Id has been related to the sylvatic cycle. In order to demonstrate that the circulating T. cruzi I isolates in Colombia can be classified in the four proposed haplotypes, specific primers were designed on polymorphic regions of the miniexon gene's intergenic sequences. This set of primers allowed the molecular characterization of 33 Colombian isolates, classifying them into three of the four proposed haplotypes (Ia, Ib and Id). Results obtained from maximum parsimony and maximum-likelihood-based phylogenetic analyses correlated with the molecular classification of the isolates and their transmission cycles. This study brings insights into the Chagas disease epidemiology and the parasite's transmission dynamics.

Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.

Variabilit&eacute; g&eacute;n&eacute;tique chez le Trypanosoma cruzi qu&#39;i groupe a r&eacute;cemment &eacute;t&eacute; r&eacute;v&eacute;l&eacute;e dans colombiens isolats provenant de l&#39;homme, les r&eacute;servoirs et les vecteurs. R&eacute;arrangements g&eacute;nomiques et les r&eacute;gions polymorphes des marqueurs taxonomiques, tel que le g&egrave;ne miniexon, ont conduit &agrave; l&#39;&eacute;laboration d&#39;outils mol&eacute;culaires pour identifier les haplotypes phylog&eacute;n&eacute;tique dans T. cruzi isolats. Des polymorphismes g&eacute;n&eacute;tiques dans T. cruzi j&#39;isole, ils ont &eacute;t&eacute; class&eacute;s en quatre haplotypes selon leurs cycles de transmission &eacute;pid&eacute;miologique. Haplotype Ia est associ&eacute; &agrave; des isolats de domestiques, de Rhodnius prolixus ; haplotype Ib, avec le cycle domestiques et p&eacute;ridomestiques, principalement associ&eacute; aux Triatoma dimidiata ; haplotype Ic est un groupe peu caract&eacute;ris&eacute;, ce qui a &eacute;t&eacute; associ&eacute; par le cycle p&eacute;ridomestiques ; et haplotype Id a &eacute;t&eacute; li&eacute; au cycle sylvatique. Afin de d&eacute;montrer que le circulant de T. cruzi j&#39;isole en Colombie peut &ecirc;tre class&eacute;e en quatre haplotypes propos&eacute;es, des amorces sp&eacute;cifiques ont &eacute;t&eacute; con&ccedil;us sur des r&eacute;gions polymorphes de s&eacute;quences interg&eacute;niques du g&egrave;ne de la miniexon. Cette s&eacute;rie d&#39;amorces a permis la caract&eacute;risation mol&eacute;culaire des 33 isolats colombiens, classifiant dans trois des quatre haplotypes propos&eacute;es (Ia, Ib et Id). R&eacute;sultats obtenus par les analyses phylog&eacute;n&eacute;tiques bas&eacute;es sur la vraisemblance-maximum de parcimonie et de corr&eacute;lation avec la classification mol&eacute;culaire des souches et leurs cycles de transmission. Cette &eacute;tude permet de mieux comprendre l&#39;&eacute;pid&eacute;miologie de la maladie de Chagas et la dynamique de la transmission du parasite.

Haplotype identification au sein de Trypanosoma cruzi, j&#39;ai en colombien isolats provenant de plusieurs r&eacute;servoirs, des vecteurs et des humains.

Variabilit&agrave; genetica nel Trypanosoma cruzi che gruppo recentemente &egrave; stato rivelato in colombiani isolati da esseri umani, serbatoi e vettori. Riarrangiamenti genomici e regioni polimorfiche marcatori tassonomico, come il gene miniexon, che hanno portato allo sviluppo di strumenti molecolari per identificare filogenetici aplotipi in T. cruzi isolati. Da polimorfismi genetici trovati in T. cruzi isolati, esse sono state classificate in quattro aplotipi secondo loro cicli di trasmissione epidemiologica. Ia aplotipo &egrave; associato con ceppi domestici, da Rhodnius prolisso; aplotipo Ib, con il ciclo domestico e peridomestic, principalmente associato Triatoma dimidiata; aplotipo Ic &egrave; un gruppo scarsamente caratterizzato, che &egrave; stato associato con il ciclo peridomestic; e aplotipo Id &egrave; stato correlato al ciclo Silvestre. Al fine di dimostrare che il T. cruzi circolanti isolati in Colombia pu&ograve; essere classificata nei quattro proposti aplotipi, primer specifici sono stati progettati su regioni polimorfiche di intergenic sequenze del gene della miniexon. Questo set di primer ha permesso la caratterizzazione molecolare di isolati colombiani 33, classificandole in tre le quattro proposti aplotipi (Ia, Ib e Id). Risultati ottenuti dalla massima parsimonia e analisi filogenetiche basate su massimo-probabilit&agrave; correlata con la classificazione molecolare dei loro cicli di trasmissione e gli isolati. Questo studio porta intuizioni l&#39;epidemiologia del morbo di Chagas e dinamiche di trasmissione del parassita.

Identificazione aplotipo all&#39;interno di Trypanosoma cruzi in colombiano isolati da diversi bacini, vettori e gli esseri umani.

Phylogenetic studies of Trypanosoma cruzi have identified the existence of two groups: T. cruzi I and T. cruzi II. There are aspects that still remain unknown about the genetic variability within the T. cruzi I group. Given its epidemiological importance, it is necessary to have a better understanding of T. cruzi transmission cycles. Our purpose was to corroborate the existence of haplotypes within the T. cruzi I group and to describe the genetic variability and phylogenetic relationships, based on single nucleotide polymorphisms (SNPs) found in the miniexon gene intergenic region, for the isolates from different hosts and epidemiological transmission cycles in Colombian regions. 31 T. cruzi isolates were molecularly characterized. Phylogenetic relationships within T. cruzi I isolates showed four haplotype groups (Ia-Id), associated with their transmission cycle. In previous studies, we reported that haplotype Ia is mainly associated with the domestic cycle and domiciliated Rhodnius prolixus. Haplotype Ib is associated with the domestic cycle and peridomestic cycle, haplotype Ic is closely related with the peridomestic cycle, and haplotype Id is strongly associated with the sylvatic cycle. The phylogenetic methodologies applied in this study are tools that bolster the associations among isolates and thus shed light on Chagas disease epidemiology.

Genetic Variability and Phylogenetic Relationships within Trypanosoma cruzi I Isolated in Colombia Based on Miniexon Gene Sequences.

Des &eacute;tudes phylog&eacute;n&eacute;tiques de Trypanosoma cruzi ont identifi&eacute; l&#39;existence de deux groupes: T. cruzi I et T. cruzi II. Il y a des aspects qui restent encore inconnues sur la variabilit&eacute; g&eacute;n&eacute;tique au sein de le T. cruzi de groupe. Compte tenu de son importance &eacute;pid&eacute;miologique, il est n&eacute;cessaire d&#39;avoir une meilleure compr&eacute;hension des cycles de transmission T. cruzi. Notre but &eacute;tait de corroborer l&#39;existence des haplotypes dans le T. cruzi de groupe et de d&eacute;crire la variabilit&eacute; g&eacute;n&eacute;tique et les relations phylog&eacute;n&eacute;tiques, bas&eacute;es sur les polymorphismes mononucl&eacute;otidiques (SNP) trouv&eacute;s dans la r&eacute;gion interg&eacute;nique miniexon g&egrave;ne, pour les isolats provenant de diff&eacute;rents h&ocirc;tes et cycles de transmission &eacute;pid&eacute;miologique dans les r&eacute;gions colombiennes. 31 T. cruzi isolats ont &eacute;t&eacute; caract&eacute;ris&eacute;s mol&eacute;culairement. Relations phylog&eacute;n&eacute;tiques au sein de T. cruzi j&#39;isolats ont montr&eacute; haplotype quatre groupes (Ia-Id), associ&eacute;es &agrave; leur cycle de transmission. Dans des &eacute;tudes pr&eacute;c&eacute;dentes, nous avons signal&eacute; que cet haplotype Ia est principalement associ&eacute; &agrave; l&#39;int&eacute;rieur cycle et domicili&eacute;e Rhodnius prolixus. Haplotype Ib est associ&eacute; au cycle domestique et p&eacute;ridomestiques cycle, haplotype Ic est &eacute;troitement li&eacute; avec le cycle p&eacute;ridomestiques, et haplotype Id est fortement li&eacute;e au cycle sylvatique. La phylog&eacute;n&eacute;tique m&eacute;thodologie employ&eacute;e dans cette &eacute;tude est des outils qui renforceront les associations entre les isolats et ainsi faire la lumi&egrave;re sur l&#39;&eacute;pid&eacute;miologie de la maladie de Chagas.

Variabilit&eacute; g&eacute;n&eacute;tique et relations phylog&eacute;n&eacute;tiques au sein de Trypanosoma cruzi j&#39;ai isol&eacute; en Colombie bas&eacute;es sur les s&eacute;quences de g&egrave;ne de Miniexon.

Studi filogenetici di Trypanosoma cruzi hanno identificato l&#39;esistenza di due gruppi: T. cruzi I e T. cruzi II. Ci sono aspetti che ancora rimangono sconosciuti circa la variabilit&agrave; genetica all&#39;interno del gruppo T. cruzi. Data la sua importanza epidemiologica, &egrave; necessario disporre di una migliore comprensione dei cicli di trasmissione di T. cruzi. Il nostro scopo era di confermano l&#39;esistenza di aplotipi all&#39;interno del gruppo T. cruzi e per descrivere la variabilit&agrave; genetica e relazioni filogenetiche, basati su polimorfismi a singolo nucleotide (SNPs) trovati nella regione intergenic di miniexon gene, per gli isolati da diversi host e cicli di trasmissione epidemiologica nelle regioni colombiane. 31 T. cruzi isolati sono stati caratterizzati molecolarmente. Relazioni filogenetiche all&#39;interno di T. cruzi isolati ha mostrato aplotipo quattro gruppi (Ia-Id), associati con il loro ciclo di trasmissione. In studi precedenti, abbiamo segnalato che tale aplotipo Ia &egrave; principalmente associato con l&#39;interno ciclo e domiciliated Rhodnius prolisso. Aplotipo Ib &egrave; associato con il ciclo interno e peridomestic ciclo, aplotipo Ic &egrave; strettamente correlato con il ciclo di peridomestic, e aplotipo Id &egrave; fortemente associato con il ciclo Silvestre. La filogenetiche metodologie applicate in questo studio sono strumenti che rafforzare le associazioni tra gli isolati e cos&igrave; far luce sulla epidemiologia di malattia di Chagas.

Variabilit&agrave; genetica e la relazioni filogenetiche all&#39;interno di Trypanosoma cruzi isolati in Colombia basato su sequenze di geni Miniexon.

Internal and geographical clustering within Trypanosoma cruzi I (TcI) has been recently revealed by using Multilocus Microsatellite Typing and sequencing of the Spliced-Leader Intergenic Region (SL-IR). In the present work, 14 isolates and 11 laboratory-cloned stocks obtained from a geographically restricted area in Chaco Province, Argentina, were analyzed by PCR and sequencing of SL-IR. We were able to differentiate 8 different genotypes that clustered into 4 groups. One of these groups was classified within the formerly described haplotype A and another one within the recently described SL-IR group E. Both were phylogenetically well-supported. In contrast, none of the stocks from the Chaco province were grouped within the cluster previously named haplotype D despite the fact that they shared a similar microsatellite motif in the SL-IR. No evidence of recombination or gene conversion within these stocks was found. On the other hand, multiple ambiguous alignments in the microsatellite region of SL-IR, affecting the tree topology and relationships among groups were detected. Finally, since there are multiple copies of the SL-IR, and they are arranged in tandem, we discuss how molecular processes affecting this kind of sequences could mislead phylogenetic inference.

Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco.

Interne et g&eacute;ographique de regroupement au sein de Trypanosoma cruzi je (TcI) a &eacute;t&eacute; r&eacute;cemment r&eacute;v&eacute;l&eacute; par &agrave; l&#39;aide de dactylographie de microsatellites &agrave; lieux multiples et le s&eacute;quen&ccedil;age de la r&eacute;gion interg&eacute;nique Spliced-Leader (SL-IR). Dans le pr&eacute;sent travail, 14 isolats et 11 stocks de laboratoire clon&eacute;s provenant d&#39;une r&eacute;gion g&eacute;ographiquement limit&eacute;e dans la Province du Chaco (Argentine), ont &eacute;t&eacute; analys&eacute;s par PCR et s&eacute;quen&ccedil;age de SL-IR Nous avons &eacute;t&eacute; en mesure de distinguer 8 g&eacute;notypes diff&eacute;rents qui forment 4 groupes. Un de ces groupes a &eacute;t&eacute; class&eacute; au sein de l&#39;haplotype anciennement d&eacute;crite A et l&#39;autre au sein du groupe r&eacute;cemment d&eacute;crit de SL-IR E. Tous deux &eacute;taient bien sur le plan phylog&eacute;n&eacute;tique. En revanche, aucun des stocks de la province de Chaco ont &eacute;t&eacute; regroup&eacute;s au sein du cluster qui se nommait auparavant haplotype D malgr&eacute; le fait qu&#39;ils partageaient un motif similaire de microsatellites dans le SL-IR. A trouv&eacute; aucune preuve de recombinaison ou g&egrave;ne de conversion au sein de ces stocks. En revanche, plusieurs alignements ambigu&euml;s dans la r&eacute;gion de microsatellites de SL-IR, affectant la topologie de l&#39;arborescence et les relations entre les groupes ont &eacute;t&eacute; d&eacute;tect&eacute;s. Enfin, puisqu&#39;il existe plusieurs copies de la SL-IR, et elles sont dispos&eacute;es en tandem, nous discutons comment mol&eacute;culaires processus affectant ce type de s&eacute;quences pourrait induire en erreur inf&eacute;rence phylog&eacute;n&eacute;tique.

Int&eacute;r&ecirc;t et limites des s&eacute;quences &eacute;piss&eacute;es Leader r&eacute;gion interg&eacute;nique pour l&#39;analyse de Trypanosoma cruzi je diversit&eacute; phylog&eacute;n&eacute;tique du Chaco argentin.

Interna e geografica clustering di Trypanosoma cruzi I (TcI) ha recentemente rivelato mediante Multilocus Microsatellite digitando e sequenziamento della regione Intergenic Spliced Leader (SL-IR). Nel presente lavoro, sono stati analizzati 14 isolati e 11 stock clonato in laboratorio, ottenuto da un&#39;area geograficamente ristretta nella provincia del Chaco, Argentina, PCR e sequenziamento di SL-IR. Siamo stati in grado di distinguere 8 diversi genotipi che raggruppati in 4 gruppi. Uno di questi gruppi &egrave; stato classificato all&#39;interno dell&#39;aplotipo precedentemente descritto A e un&#39;altra all&#39;interno del gruppo SL-IR recentemente descritto E. Entrambi erano filogeneticamente ben supportati. Al contrario, nessuno degli stock della provincia del Chaco sono stati raggruppati all&#39;interno del cluster precedentemente denominato aplotipo D nonostante il fatto che hanno condiviso un simile motivo microsatellite in SL-IR. &Egrave; stata trovata alcuna prova di conversione ricombinazione o gene all&#39;interno di questi stock. D&#39;altra parte, sono stati individuati allineamenti multipli di ambigui nella regione microsatellite di SL-IR, che colpisce l&#39;albero filogenetico e le relazioni tra gruppi. Infine, poich&eacute; ci sono pi&ugrave; copie di SL-IR, e sono disposti in tandem, discutiamo come molecolari dei processi che interessano questo tipo di sequenze potrebbe trarre in inganno l&#39;inferenza filogenetica.

Interesse e le limitazioni delle sequenze Spliced Leader Intergenic regione per analizzare il Trypanosoma cruzi diversit&agrave; filogenetica del Chaco argentino.

Type II Toxoplasma gondii KU80 knockouts (Î”ku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Î”ku80 Î”hxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Î”ku80 Î”hxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Î”gra4 and Î”gra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Î”ku80 Î”hxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.

Type II Toxoplasma gondii KU80 d&eacute;bouchures (&Delta;ku80) d&eacute;ficientes en fin non-homologues rejoindre ont &eacute;t&eacute; d&eacute;velopp&eacute;s pour supprimer le trajet dominant m&eacute;diation int&eacute;gration al&eacute;atoire du ciblage des &eacute;pisomes. Ciblage de fr&eacute;quence dans la souche du type II &Delta;ku80 &Delta;hxgprt g&eacute;nique mesur&eacute;e &agrave; l&#39;orotate (FCT) et les loci de phosphoribosyltransf&eacute;rase uracile (UPRT) a &eacute;t&eacute; tr&egrave;s efficace. Pour &eacute;valuer le potentiel de la souche &Delta;hxgprt de type II &Delta;ku80 pour &eacute;tudier la fonction des g&egrave;nes affectant la biologie du kyste et stades latentes de l&#39;infection, nous avons cibl&eacute; la suppression de quatre g&egrave;nes antig&egrave;ne parasite (GRA4, GRA6, ROP7 et tgd057) qui codent caract&eacute;ris&eacute;es &eacute;pitopes T CD8 qui d&eacute;clenchent les populations correspondantes sp&eacute;cifiques de l&#39;antig&egrave;ne CD8 T cell associ&eacute;es au contr&ocirc;le de l&#39;infection. D&eacute;veloppement du kyste dans ces souches mutantes de type II n&#39;a pas &eacute;t&eacute; trouv&eacute; strictement d&eacute;pendante de l&#39;antig&egrave;ne-sp&eacute;cifiques CD8 T cell h6te. En revanche, un r&ocirc;le biologique important a &eacute;t&eacute; r&eacute;v&eacute;l&eacute; pour les prot&eacute;ines des granules denses GRA4 et GRA6 dans le d&eacute;veloppement de kystes puisque les charges de cerveau tissu kyste ont &eacute;t&eacute; r&eacute;duites drastiquement sp&eacute;cifiquement chez les souches mutantes avec GRA4 et/ou GRA6 supprim&eacute;. Compl&eacute;mentation des souches mutantes &Delta;gra4 et &Delta;gra6 &agrave; l&#39;aide d&#39;un all&egrave;le fonctionnel de la GRA supprim&eacute; codage r&eacute;gion plac&eacute;e sous le contr&ocirc;le du locus UPRT endog&egrave;ne s&#39;est av&eacute;r&eacute;e pour significativement restaurer fardeaux de kyste de cerveau. Ces r&eacute;sultats r&eacute;v&egrave;lent que les prot&eacute;ines GRA jouent un r&ocirc;le fonctionnel dans l&#39;&eacute;tablissement des charges de kyste et une infection latente. Collectivement, nos r&eacute;sultats sugg&egrave;rent qu&#39;un type de bagage g&eacute;n&eacute;tique de la &Delta;ku80 de &Delta;hxgprt II permet une analyse fonctionnelle des d&eacute;bits plus &eacute;lev&eacute;s du g&eacute;nome du parasite, de r&eacute;v&eacute;ler des aspects fondamentaux de la biologie de parasite contr&ocirc;lant la virulence, la pathogen&egrave;se et la transmission.

Souches de type II Toxoplasma gondii KU80 knockout permettent une analyse fonctionnelle des g&egrave;nes n&eacute;cessaires pour le d&eacute;veloppement de kystes et d&#39;une infection latente.

Tipo II Toxoplasma gondii KU80 knockouts (&Delta;ku80) carenti di unirsi nonhomologous fine sono stati sviluppati per eliminare la dominante via mediazione integrazione casuale di targeting episomes. Gene targeting per frequenza nel tipo II &Delta;ku80 &Delta;hxgprt ceppo misurata presso l&#39;orotato (= OPRT) e i loci di adenosin fosforibosiltrasferasi uracile (UPRT) era altamente efficiente. Per valutare il potenziale del ceppo tipo II &Delta;ku80 &Delta;hxgprt per esaminare la funzione del gene che interessa biologia cisti e stadi latente dell&#39;infezione, abbiamo mirato l&#39;eliminazione di quattro parassita antigene geni (GRA4, GRA6, ROP7 e tgd057) che codificano caratterizzati cellule T cell epitopi che suscitano corrispondente antigene-specifiche cellule T popolazioni di cellule associate al controllo dell&#39;infezione. Lo sviluppo di cisti in questi ceppi mutanti di tipo II non &egrave; stato trovato per essere strettamente dipendente da risposte antigene-specifiche cellule T cell host. Al contrario, un significativo ruolo biologico &egrave; stato rivelato per le proteine dei granuli densi GRA4 e GRA6 nello sviluppo di cisti poich&eacute; gli oneri di cisti tessuto cerebrale sono stati drasticamente ridotta in particolare ceppi mutanti con GRA4 o GRA6 cancellato. Complementazione dei ceppi mutanti &Delta;gra4 e &Delta;gra6 utilizzando un allele funzionale del GRA eliminato codifica regione posizionato sotto il controllo del locus UPRT endogeno &egrave; stato trovato per ripristinare in modo significativo gli oneri di cisti cerebrali. Questi risultati rivelano che le proteine GRA giocano un ruolo funzionale nella creazione di cisti oneri e infezione latente. Collettivamente, i nostri risultati suggeriscono che un tipo II &Delta;ku80 &Delta;hxgprt fondo genetico permette una velocit&agrave; superiore di analisi funzionale del genoma del parassita per rivelare gli aspetti fondamentali della biologia del parassita controllo virulenza, patogenesi e trasmissione.

Tipo II Toxoplasma gondii KU80 knockout ceppi consentono analisi funzionale dei geni necessari per lo sviluppo di cisti e di infezione latente.

To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern).

First Colombian multicentric newborn screening for congenital toxoplasmosis.

Pour d&eacute;terminer l&#39;incidence de la toxoplasmose cong&eacute;nitale chez le nouveau-n&eacute; colombienne de 19 h&ocirc;pital ou de la sant&eacute; maternelle et infantile, services de sept villes diff&eacute;rentes des cinq r&eacute;gions g&eacute;ographiques naturelles (Cara&iuml;bes, centrale, Andes, Amazonie et orientale).

Premier colombien multicentrique newborn screening pour toxoplasmose cong&eacute;nitale.

Per determinare l&#39;incidenza della toxoplasmosi congenita nei neonati colombiani da 19 ospedale o materna child health services da sette citt&agrave; diverse di cinque regioni geografiche naturali (Caraibi, centrale, andina, Amazzonia e orientale).

Primo colombiano multicentriche screening neonatale per la toxoplasmosi congenita.

&Eacute;piss&eacute; chef r&eacute;gion interg&eacute;nique (SL-IR) s&eacute;quences de 23 Trypanosoma rangeli souches isolement de glandes salivaires de Rhodnius colombiensis, r. ecuadoriensis, r. pallescens et r. prolixus et deux souches humaines ont r&eacute;v&eacute;l&eacute; l&#39;existence de 4 g&eacute;notypes avec CA, GT, TA, ATT et GTAUX microsatellite r&eacute;p&eacute;titions et la pr&eacute;sence d&#39;insertions/d&eacute;l&eacute;tions (INDEL) et polymorphisme de nucl&eacute;otides simples (SNP) qui caract&eacute;risent chaque g&eacute;notype. Les souches isolement de la m&ecirc;me esp&egrave;ce de vecteur ou de la m&ecirc;me lign&eacute;e &eacute;volutive Rhodnius pr&eacute;sent&eacute; les m&ecirc;mes g&eacute;notypes, m&ecirc;me dans les cas o&ugrave; les souches avaient &eacute;t&eacute; isol&eacute;es de vecteurs captur&eacute;s dans des r&eacute;gions g&eacute;ographiquement &eacute;loign&eacute;es. Le dendrogramme construit &agrave; partir des s&eacute;quences de SL-IR s&eacute;par&eacute;s chacun d&#39;eux en deux groupes principaux, l&#39;un avec les g&eacute;notypes isol&eacute;es de r. prolixus et l&#39;autre groupe contenant trois sous-groupes bien d&eacute;finis avec les g&eacute;notypes isol&eacute;es de r. pallescens, r. colombiensis et r. ecuadoriensis. Analyse de l&#39;ADN (RAPD) polymorphe amplifi&eacute; al&eacute;atoire a montr&eacute; le m&ecirc;mes deux groupes principaux et sous-groupes soutien association stricte T. rangeli g&eacute;notypes avec Rhodnius esp&egrave;ces. Combin&eacute; avec d&#39;autres &eacute;tudes, ces r&eacute;sultats sugg&egrave;rent une possible association co-&eacute;volutionnaire entre T. rangeli g&eacute;notypes et de leurs vecteurs.

Analyse de la s&eacute;quence de la r&eacute;gion interg&eacute;nique &eacute;piss&eacute;-chef (SL-IR) et al&eacute;atoire ADN polymorphe amplifi&eacute; (RAPD) de Trypanosoma rangeli souches isolement de Rhodnius ecuadoriensis, r. colombiensis, r. pallescens et r. prolixus sugg&egrave;re un certain degr&eacute; de co-&eacute;volution entre les parasites et les vecteurs.

Impiombato leader intergenic regione (SL-IR) sequenze da 23 Trypanosoma rangeli ceppi isolati dalle ghiandole salivari di Rhodnius colombiensis, r. ecuadoriensis, pallescens r. e r. prolisso e due ceppi umani ha rivelato l&#39;esistenza di 4 genotipi con ripetizioni microsatellite CA, GT, TA, ATT e GTAT e la presenza di inserzioni/delezioni (INDEL) e polimorfismo a singolo nucleotide (SNP) che caratterizzano ogni genotipo. I ceppi isolati dalla stessa specie vettore o la stessa linea evolutiva Rhodnius presentato i genotipi stessi, anche nei casi dove i ceppi erano stati isolati da vettori catturati nelle regioni geograficamente distanti. Il dendrogramma costruito dalle sequenze SL-IR separati tutti loro in due gruppi principali, uno con i genotipi isolati da r. prolisso e l&#39;altro gruppo contenente tre sottogruppi ben definiti con i genotipi isolati da pallescens r., r. colombiensis e r. ecuadoriensis. Analisi di DNA (RAPD) polimorfico amplificato casuale ha mostrato la stessi due principali gruppi e sottogruppi sostegno associazione rigorosa T. rangeli genotipi con Rhodnius specie. Combinato con altri studi, questi risultati suggeriscono una possibile associazione co-evolutiva tra T. rangeli genotipi e dei loro vettori.

Analisi di sequenza della regione intergenic spliced leader (SL-IR) e casuale amplificato polimorfici del DNA (RAPD) di Trypanosoma rangeli ceppi isolati da Rhodnius ecuadoriensis, r. colombiensis, r. pallescens e prolisso r. suggerisce un certo grado di co-evoluzione tra vettori e parassiti.

Spliced leader intergenic region (SL-IR) sequences from 23 Trypanosoma rangeli strains isolated from the salivary glands of Rhodnius colombiensis, R. ecuadoriensis, R. pallescens and R. prolixus and two human strains revealed the existence of 4 genotypes with CA, GT, TA, ATT and GTAT microsatellite repeats and the presence of insertions/deletions (INDEL) and single nucleotide polymorphism (SNP) characterizing each genotype. The strains isolated from the same vector species or the same Rhodnius evolutionary line presented the same genotypes, even in cases where strains had been isolated from vectors captured in geographically distant regions. The dendrogram constructed from the SL-IR sequences separated all of them into two main groups, one with the genotypes isolated from R. prolixus and the other group containing three well defined sub-groups with the genotypes isolated from R. pallescens, R. colombiensis and R. ecuadoriensis. Random amplified polymorphic DNA (RAPD) analysis showed the same two main groups and sub-groups supporting strict T. rangeli genotypes' association with Rhodnius species. Combined with other studies, these results suggest a possible co-evolutionary association between T. rangeli genotypes and their vectors.

Sequence analysis of the spliced-leader intergenic region (SL-IR) and random amplified polymorphic DNA (RAPD) of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis, R. colombiensis, R. pallescens and R. prolixus suggests a degree of co-evolution between parasites and vectors.

Article	Year
Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans. Acta tropica\| PubMed ID: 19135020	2009
Genetic Variability and Phylogenetic Relationships within Trypanosoma cruzi I Isolated in Colombia Based on Miniexon Gene Sequences. Journal of parasitology research\| PubMed ID: 20798881	2009
Interest and limitations of Spliced Leader Intergenic Region sequences for analyzing Trypanosoma cruzi I phylogenetic diversity in the Argentinean Chaco. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 21111067	2011
Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryotic cell\| PubMed ID: 21531875	2011
First Colombian multicentric newborn screening for congenital toxoplasmosis. PLoS neglected tropical diseases\| PubMed ID: 21655304	2011
Sequence analysis of the spliced-leader intergenic region (SL-IR) and random amplified polymorphic DNA (RAPD) of Trypanosoma rangeli strains isolated from Rhodnius ecuadoriensis, R. colombie... Acta tropica\| PubMed ID: 21718675	2011

Alejandra Falla

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