Rattini (Rodentia, Murinae) species relationships and involvement as reservoirs for Scrub Typhus : a comparative molecular cytogenetic gene expression approach

Badenhorst, Daleen (2011-12)

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: The Rattus sensu lato complex, which harbours the important biomedical model species Rattus norvegicus, represents a group of rodents that are of clinical, agricultural and epidemiological importance. This study presents a comprehensive comparative molecular cytogenetic investigation of this complex. Karyotypes of 11 Rattus s. l. species and Hapalomys delacouri were analysed using conventional cytogenetic techniques. These data suggest that, contrary to previous claims, the usefulness of karyotypes for identifying these Asian murid species is limited, as few species-specific chromosomal characters could be identified. In order to understand the events that shaped the morphology and composition of the extant karyotypes, nine out of the 11 Rattus s. l. species were analysed by chromosome painting. This allowed the detection of a rare convergent sex-autosome translocation present in H. delacouri and B. savilei, whose establishment is hypothesised to have been favoured by the presence of interstitial heterochromatic blocks (IHBs) at the boundaries of the translocated segments. These results indicate that Rattus s. l. is characterised by slow to moderate rates of chromosome evolution in contrast to the extensive chromosome restructuring identified in most other murid rodents. Based on these data the first comprehensive putative Rattini ancestral karyotype was constructed. Their integration with published comparative maps enabled a revision of the previously postulated ancestral murid karyotype. BAC-mapping unequivocally demonstrated that the widely reported polymorphisms affecting chromosome pairs 1, 12 and 13 in the Rattus karyotypes are due to pericentric inversions. The analysis of genomic features, such as telomeres, Ag-NORs and satellite DNA suggest a constrained pattern of chromosome evolution. The investigated rat satellite I DNA family appears to be taxon (Rattus) specific, and of recent origin (consistent with a feedback model of satellite evolution). The comparative nature of the study led to the further analysis of the species within Rattus s. l. complex and their possible involvement as reservoirs of scrub typhus using a qPCR gene expression approach based on real-time PCR. The structure and transcription of syndecan-4, which had previously been linked to Orientia tsutsugamushi (causative agent of scrub typhus) infection, was compared among Rattini (typhus-positive) and Murini (typhus-negative) rodents. Although, it was not possible to conclusively link the structural variation observed between Rattini and Murini with carrier status, a link was identified between underexpression of syndecan-4 in Murini and seropositive Rattini rodents, compared to those that were seronegative. This suggests that the reduced levels of syndecan-4 transcription in Murini and Rattini is linked to the poor carrier status of Murini, and to increased longevity of seropositive Rattini (i.e., predominant host of Orientia), highlighting aspects that need further investigation.

AFRIKAANSE OPSOMMING: Die Rattus sensu lato kompleks, wat die belangrike biomediese model spesie Rattus norvegicus huisves, bevat ‘n groep knaagdiere wat van kliniese, landbou, en epidiomologiese belang is. Hierdie studie bevat ‘n omvattende sitogenetiese molekulêre vergelyking binne hierdie kompleks. Die kariotipes van 11 Rattus s. l. spesies en Hapalomys delacouri is ondersoek met die gebruik van konvensionele sitogenetiese tegnieke. Die data dui daarop, in kontras met vorige bevindinge, dat die bruikbaarheid van kariotipes om Asiese knaagdiere te identifiseer beperk is, aangesien min spesies-spesifieke chromosoom merkers geidentifiseer kon word. Om die gebeure wat tot die morfologie en komposisie van die huidige kariotipes gelei het, te verstaan, is nege van die 11 Rattus s. l. spesies met behulp van chromosoom fluoressente hibridisasie ondersoek. Dit het die ontdekking van ‘n rare konvergente geslagschromosoom-outosoom translokasie in H. delacouri en B. savilei tot gevolg gehad. Die vaslegging hiervan is heel moontlik bevoordeel deur die teenwoordigheid van interkalerende heterchromatien blokke (IHBs) by die grens van die translokeerde segmente. Hierdie resultate dui daarop dat Rattus s. l. deur ‘n stadige tempo van chromosoom verandering gekenmerk word, wat in skrille kontras staan met die hoë aantal chromosoom herrangskikkings wat in meeste ander murid knaagdiere geidentifiseer is. Vanuit hierdie data kon die eerste oerouer kariotipe van die Rattini bepaal word. Die gebruik van hierdie data in kombinasie met beskikbare vergelykende kaarte het dit moontlik gemaak om die vorige hipotetiese oerouer murid kariotipe te hersien. BAC-kartering het dit moontlik gemaak om sonder twyfel vas te stel dat die polimorfisme wat chromosoom pare 1, 12 en 13 in die Rattus kariotipe affekteer, die resultaat van perisentriese omsetting is. Die analise van genomiese eienskappe, soos telomere, Ag-NORs en satelliet DNA dui op ‘n beperkte patroon van chromosoom evolusie. Die bestudeerde rot satelliet DNA familie blyk takson (Rattus) spesifiek te wees met ‘n onlangse oorsprong, wat ooreenstem met die terugvoer model van satellite-evolusie. Die vergelykende aard van hierdie studie het gelei tot die verdere analise van die spesies in die Rattus s. l. kompleks in terme van hul moontlike rol as draers van “scrub typhus” deur gebruik te maak van qPCR geen uitdrukking, wat gebasseer is op “real-time” PCR. Die struktuur en transkripsie van syndecan-4, wat in die verlede aan Orientia tsutsugamushi (veroorsakende agent van scrub typhus) infeksie gekoppel is, is tussen Rattini (typhuspositief) en Murini (typhus-negatief) knaagdiere vergelyk. Ten spyte daarvan dat dit nie moontlik was om die strukturele variasie tussen Rattini en Murini met draer status te koppel nie, is daar wel ‘n skakel tussen die verlaagde uitdrukking van syndecan-4 in Murini en seropositiewe Rattini knaagdiere, in vergelyking met die wat sero-negatief was, gevind. Dit stel voor dat die verlaagde vlakke van syndecan-4 transkripsie in Murini en Rattini aan die swak draer status van Murini, asook die verhoogde langslewendheid van sero-positiewe Rattini (i.e., oorheersende gasheer van Orientia), gekoppel is. Hierdie bevindinge beklemtoon sekere belangrike aspekte vir verdere navorsing.

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