Serološka i molekularna tipizacija sojeva leptospira izdvojenih iz mišolikih glodavaca

master's thesis

• Title (english): Serological and Molecular Typization of Leptospira spp. Isolated from Small Rodents
• Author: Luka Crnjac
• Mentor: Josipa Habuš (mentor)
• Committee member: Josipa Habuš (predsjednik povjerenstva)
• Committee member: Zoran Milas (član povjerenstva)
• Committee member: Zrinka Štritof (član povjerenstva)
• Granter: University of Zagreb; Faculty of Veterinary Medicine; (Department of Microbiology and Infectious Diseases with Clinic); Zagreb
• Defense date and country: 2020-02-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Veterinary Clinical Sciences

Abstract

Leptospiroza je (re)emergentna zoonoza uzrokovana izrazito heterogenim bakterijama iz roda Leptospira. Rod Leptospira se na osnovu genotipskih i fenotipskih razlika moţe podijeliti na 22 genomske vrste i više od 320 saprofitskih, intermedijarnih i patogenih serovara. Svi patogeni serovari mogu zaraziti više vrsta domaćih i divljih ţivotinja i ĉovjeka, no u pojedinom podruĉju kruţe zahvaljujući odreĊenoj ţivotinjskoj vrsti koja predstavlja rezervoar tog serovara. Neka od prijašnjih istraţivanja utvrdila su odreĊene osobitosti leptospiroze kod ljudi u Republici Hrvatskoj, kao što su visoka prevalencija bolesti u ljudi i pojava vjerojatno infektivnih serovara ĉiji su rezervoari mišoliki glodavci. Navedeno ĉini Hrvatsku drţavom sa najviše sluĉajeva leptospiroze u ljudi u Europskoj Uniji, s obzirom na broj stanovnika, odnosno trinaestom u svijetu. Slijedom navedenog, ciljevi ovog istraţivanja bili su serološki i molekularno tipizirati sojeva leptospire izdvojenih iz mišolikih glodavaca izlovljenih na podruĉju prirodnih ţarišta Republike Hrvatske, te utvrditi povezanosti odreĊenih vrsta mišolikih glodavaca s pojedinim serovarima Leptospira.
Tijekom ovog istraţivanja pretraţeni su svi izolati (n=101) leptospira izdvojeni iz mišolikih glodavaca i pohranjeni u arhivu Laboratorija za leptospire, Zavoda za mikrobiologiju i zarazne bolesti s klinikom, Veterinarskog fakulteta, Sveuĉilišta u Zagrebu. Mikroskopskom aglutinacijom pomoću 12 hiperimunih seruma proizvedenih na kunićima utvrĊena je pripadnost nedeterminiranih izolata serološkim skupinama Pomona, Australis, Grippotyphosa i Bataviae. Zatim su odabrana 33 izolata koja su dalje tipizirana i molekularnim metodama: umnaţanjem secY gena, sekvencioniranjem i filogenijskom analizom odsjeĉaka, kako bi se utvrdila genomska vrsta. Za kraj je odabrano 11 izolata koji su predstavljali reprezentativan uzorak molekularno i serološki pretraţenih izolata koji su determinirani upotrebom molekularne metode tipizacije na osnovu multilokusnih sekvenci (MLST).
Dobiveni rezultati pokazali su veliku genetsku srodnost pretraţenih izolata unutar pojedinih genomskih vrsta i time potvrdili teoriju o postojanju arhaiĉnih prirodnih ţarišta, a primjećena je i povezanost pojedinih serovara sa odreĊenim vrstama mišolikih glodavaca.

Abstract (english)

Leptospirosis is one of the most widespread (re)emergent zoonoses caused by a highly heterogeneous genus Leptospira. Based on genotypic and phenotypic differences, Leptospira can be divided into 22 genomospecies and more than 320 saprophytic, intermediate and pathogenic serovars. All pathogenic serovars can infect multiple species of domestic and wild animals as well as humans. However, every serovar has its own predilection host that is considered to be its main reservoir in that specific geographical area. Some of the previous studies of human leptospirosis in Croatia revealed some specificity, primarily concerning the high incidence and prevalence of presumptive infective serogroups carried by small rodents. This makes Croatia the country with the highest incidence of human leptospirosis cases in Europan Union, in terms of population and thirteen in the world.
The aim of this study was to type strains of Leptospira isolated from small rodents using a serological and molecular techinques and to determine the association of certain rodent species with a particular serovar of Leptospira.
In this study, all Leptospira strains isolated from small rodents (n=101) during the past years and stored in the archives of the Department of Microbiology and Infectious Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb. Serologicaly undetermined isolates were tested using rabbit hyperimmune sera and assigned to serogroups Pomona, Australis, Grippotyphosa and Bataviae. Afterwards, representatives of serologically screened isolates (n=33) were selected for PCR based amplification of the secY gene, sequencing and phylogenetic analysis. In the end, multilocus sequence typing (MLST) method was employed in order to type and compare relationship of 11 selected strains.
Results obtained in this study showed increased genetic relatedness of the tested isolates within individual genomic species, thus confirming the theory of archaic nautural foci. In addition, connection of particular serovars with a certain species of small rodents were also noted.

• Keywords: Leptospira; mišoliki glodavci; prirodno ţarište; serološka i molekularna determinacija; MLST
• Keywords (english): Leptospira; small rodents; natural focus; serological group; genetic affinities; MLST
• Language: croatian
• URN:NBN: urn:nbn:hr:178:702194
• Study programme: Title: Veterinary Medicine; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica veterinarske medicine (doktor/doktorica veterinarske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-11-12 08:43:43