Универзитет у Београду, Факултет ветеринарске медицине
Abstract
Bruceloza pasa je bolest poznata preko četiri decenije, ali i pored toga ne postoje
standardizovani dijagnostički protokoli, kao ni generalni dogovor o najprikladnijem
dijagnostičkom testu. Svaka laboratorija definiše sopstvene kriterijume. Ovakva
raznovrsnost testova i nedostatak jasno definisanih protokola dovodi do teškoća u
interpretaciji rezultata seroloških testova u različitim laboratorijama. Iz tog razloga, cilj
ove doktorske disertacije obuhvatio je unapređenje dijagnostike primenom preporučenih
i novih, sopstveno pripremljenih testova. Jedan od zadataka ove disertacije je bilo i
ispitivanje upotrebljivosti Bruce-ladder multiplex PCR metode za ispitivanje kliničkih
uzoraka odnosno tkiva uterusa i testisa pasa.
Sakupljen je materijal (krv, testisi i materice) od 225 nevlasničkih pasa i to 145
ženki i 80 mužjaka. Dobijeni rezultati su pokazali da je od ukupno 225 ispitanih
uzoraka, 33 ili 14,67% krvnih seruma imalo merljiv titar antitela u 2-ME TAT. Najniži
ispitivani titar od 1/50 imalo je 13 krvnih seruma ili 5,78%, kod 8 krvnih seruma
ustanovljen je titar od 1/100 ili 3,55%, dok je titar od 1/200 imalo 12 uzoraka seruma ili
5,33%. Primenom klasičnih bakterioloških metoda, B. canis je izolovana iz tri uzorka
homogenizata tkiva reproduktivnih organa (1,33%) i to iz 2 uzorka poreklom od
mužjaka i jednog uzorka poreklom od ženke. Jedan izolat je poticao od serološki
negativnog psa. Od 225 uzoraka ispitanih Bruce-ladder multiplex PCR metodom,
pozitivna reakcija je ustanovljena kod dva (0,88%). Pre formulacije indirektnog ELISA
testa, određivane su koncentracije proteina. Elektroforetska analiza antigena dobijenih
toplotom i ultrazvukom kao i denzitometrijska kvantifikacija su pokazale da je u
antigenu dobijenom toplotom, najzastupljenija frakcija molekulske mase 10,95 kDa sa
učešćem od čak 43,12% koja odgovara R-LPS-u brucela. Ista frakcija je u antigenu
dobijenom ultrazvukom bila zastupljena sa 11,56%, odnosno u količini koja je bila 3,7x manja...The scientific community has known about canine brucellosis for over four
decades, even so, there are no standardized diagnostic protocols, nor a general
agreement on the most appropriate diagnostic test. Each laboratory defines its own
criteria. This variety of tests and the lack of clearly defined protocols lead to difficulties
in interpreting the results of serological tests in different laboratories. For this reason,
the goal of this doctoral dissertation has been to improve diagnostics using
recommended and new, self-prepared tests. One of the tasks of this dissertation was to
examine the usability of the Bruce-ladder multiplex PCR method for testing clinical
samples, i.e. dog uterine and testicular tissues.
The material (blood, testicles and uteruses) was collected from 225 dogs without
owners, 145 female and 80 male dogs. The results obtained showed that from a total of
225 tested samples, 33 or 14,67% of blood sera had measurable antibody titer in 2-ME
TAT. 13 or 5,78% blood sera had the lowest tested titer of 1/50, in 8 or 3,55% blood
sera a titer of 1/100 was determined, while 12 serum samples or 5,33% had the titer of
1/200. By applying classic bacteriological methods, B. canis was isolated from three
samples of homogenized reproductive organ tissue (1,33%), 2 from samples originating
from males and one specimen originating from a female. One isolate originated from a
serologically negative dog. Of the 225 samples assayed using the Bruce-ladder
multiplex PCR method, a positive reaction was established in two (0,88%). Protein
concentrations were determined prior to the formulation of the indirect ELISA test. The
electrophoretic analysis of antigens retrieved using heat and ultrasound, as well as the
densitometric quantification, showed that the antigen retrieved using heat had the most
prevalent molecular weight fraction of 10,95 kDa with a participation of as much as
43,12% that corresponds to Brucella R LPS. The same fraction was represented in the
antigen retrieved using ultrasound with 11,56%, or in a quantity that was 3,7 times
lower..