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In September 2018, a four-month-old dog with fever and petechial bleeding came to the internal clinic at the Faculty of Veterinary Medicine University of Belgrade. On hematology analysis, thrombocytopenia and mild anemia were observed. Examination of the blood smear revealed platelet inclusions. The commercial serology test was positive for Anaplasma spp. The dog was treated with doxycycline for 14 days, and after 48 hours from the beginning of the treatment, the symptoms subsided. PCR analysis and sequencing confirmed infection with A. platys. U septembru 2018. godine, štene staro četiri meseca, sa znacima groznice i petehijalnim krvarenjem, je došlo u ambulantu Klinike za male životinje Fakulteta veterinarske medicine Univerziteta u Beogradu. Hematološka analiza je pokazala trombocitopeniju i blagu anemiju. Pregledom krvnog razmaza ustanovljene su inkluzije u trombocitima. Komercijalni serološki test je pokazao prisustvo antitela protiv Anaplasma spp. Sprovedena je terapija doksiciklinom u trajanju od 14 dana, a posle 48 sati od početka terapije znaci bolesti su se povukli. PCR analizom i senkvencioniranjem je potvrđeno prisustvo Anaplasma platys.

U ovoj doktorskoj disertaciji ispitano je prisustvo uzročnika kju groznice - bakterije Coxiella burnetii (C. burnetii) kod nevlasničkih pasa poreklom sa teritorije grada Beograda, primenom molekularnih i seroloških metoda. Molekularni metod je korišćen za otkrivanje uzročnika u reproduktivnim tkivima nevlasničkih pasa, kao i u krpeljima prisutnim na ispitivanim životinjama, dok je imunoenzimskim testom utvrđeno prisustvo specifičnog serološkog odgovora kod ispitivanih životinja. Za otkrivanje prisustva DNK C. burnetii u uzrocima korišćena je lančana reakcija polimeraze (Trans-PCR) kojom se umnožava IS1111 fragment C. burnetii, dok je za otkrivanje specifičnih antitela protiv C. burnetii u serumima ispitivanih pasa korišćen modifikovani komercijalni imunoenzimski test (ELISA). U istraživanju je sakupljeno 316 krpelja sa 51 nevlasničkog psa i identifikovane su tri vrste krpelja: Rhipicephalus sanguineus, Ixodes ricinus i Dermacentor reticulatus. Prisustvo DNK C. burnetii ustanovljeno je u 10,53% (24/228) krpelja vrste R. sanguineus, koji su uzorkovani sa sedam pasa. Reproduktivna tkiva pasa sakupljena su od ukupno 105 pasa - 74 ženke (70,48%) i 31 mužjaka (29,52%). DNK C. burentii je ustanovljena kod 20,95% (22/105) pasa i to u 16,13% (5/31) uzoraka semenika pasa, odnosno u 22,97% (17/74) uzoraka materica i jajnika. Kod 29,52% (31/105) pasa je 5 ustanovljeno prisustvo specifičnih antitela protiv C. burnetii i to 32,26% (10/31) mužjaka i 28,38% (21/74) ispitanih ženki... In this doctoral dissertation, the presence of the causative agent of Q fever - the bacterium Coxiella burnetii (C. burnetii) in stray dogs has been examined using molecular and serological methods. Molecular method was used for the detection of the agent in the reproductive tissues of stray dogs, as well as in the ticks recovered from the examined animals, while immunoenzyme test was used for the detection of specific serological response in the examined animals. A polymerase chain reaction (Trans-PCR) targeting IS1111 element of C. burnetii was used for the detection of C. burnetii DNA in the samples, while a modified commercial immunoassay (ELISA) was used for the detection of specific antibodies against C. burnetii in the sera of the examined dogs. In this study, 316 ticks were recovered from 51 stray dogs and three tick species were identified: Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus. The presence of C. burnetii DNA was established in 10.53% (228/24) R. sanguineus ticks, which originated from seven dogs. Reproductive tissues of dogs were collected from a total of 105 dogs - 74 females (70.48%) and 31 males (29.52%). The presence of C. burentii DNA has been detected in 20.95% (22/105) dogs. C. burnetii DNA was detected in 7 16.13% (5/31) samples of dog testicles and in 22.97% (17/74) samples of uteri and ovaries...

In this doctoral dissertation, the presence of the causative agent of Q fever - the bacterium Coxiella burnetii (C. burnetii) in stray dogs has been examined using molecular and serological methods. Molecular method was used for the detection of the agent in the reproductive tissues of stray dogs, as well as in the ticks recovered from the examined animals, while immunoenzyme test was used for the detection of specific serological response in the examined animals. A polymerase chain reaction (Trans-PCR) targeting IS1111 element of C. burnetii was used for the detection of C. burnetii DNA in the samples, while a modified commercial immunoassay (ELISA) was used for the detection of specific antibodies against C. burnetii in the sera of the examined dogs. In this study, 316 ticks were recovered from 51 stray dogs and three tick species were identified: Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus. The presence of C. burnetii DNA was established in 10.53% (228/24) R. sanguineus ticks, which originated from seven dogs. Reproductive tissues of dogs were collected from a total of 105 dogs - 74 females (70.48%) and 31 males (29.52%). The presence of C. burentii DNA has been detected in 20.95% (22/105) dogs. C. burnetii DNA was detected in 7 16.13% (5/31) samples of dog testicles and in 22.97% (17/74) samples of uteri and ovaries... U ovoj doktorskoj disertaciji ispitano je prisustvo uzročnika kju groznice - bakterije Coxiella burnetii (C. burnetii) kod nevlasničkih pasa poreklom sa teritorije grada Beograda, primenom molekularnih i seroloških metoda. Molekularni metod je korišćen za otkrivanje uzročnika u reproduktivnim tkivima nevlasničkih pasa, kao i u krpeljima prisutnim na ispitivanim životinjama, dok je imunoenzimskim testom utvrđeno prisustvo specifičnog serološkog odgovora kod ispitivanih životinja. Za otkrivanje prisustva DNK C. burnetii u uzrocima korišćena je lančana reakcija polimeraze (Trans-PCR) kojom se umnožava IS1111 fragment C. burnetii, dok je za otkrivanje specifičnih antitela protiv C. burnetii u serumima ispitivanih pasa korišćen modifikovani komercijalni imunoenzimski test (ELISA). U istraživanju je sakupljeno 316 krpelja sa 51 nevlasničkog psa i identifikovane su tri vrste krpelja: Rhipicephalus sanguineus, Ixodes ricinus i Dermacentor reticulatus. Prisustvo DNK C. burnetii ustanovljeno je u 10,53% (24/228) krpelja vrste R. sanguineus, koji su uzorkovani sa sedam pasa. Reproduktivna tkiva pasa sakupljena su od ukupno 105 pasa - 74 ženke (70,48%) i 31 mužjaka (29,52%). DNK C. burentii je ustanovljena kod 20,95% (22/105) pasa i to u 16,13% (5/31) uzoraka semenika pasa, odnosno u 22,97% (17/74) uzoraka materica i jajnika. Kod 29,52% (31/105) pasa je 5 ustanovljeno prisustvo specifičnih antitela protiv C. burnetii i to 32,26% (10/31) mužjaka i 28,38% (21/74) ispitanih ženki...

Salmonele su najčešći prouzrokovači.crevnih infekcija kod ljudi i životinja Široko su rasprostranjene u prirodi a mogu kolonizovati različite domaćine Poebno su značajne one životinje čije se meso koristi za ishranu ljudi, a koje su i glavni izvor infekcije ljudi. Inficirane životinje ne ispoljavaju simptome i terapija se sprovodi nalazom salmonela prilikom rutinske kontrole zdravstvenog stanja. Posebno, kod ljudi i životinja salmoneloze su značajne i zbog kliconoštva koje može trajati veoma dugo zavisno od starosti inficiranog organizma. Čest su uzrok alimentarnih toksiinfekcija kod ljudi. Duž čitavog lanca ishrane moguća je i sekundarna kontaminacija salmonelama. Pored rutinskih mikrobioloških analiza u otkrivanju izvora i puteva širenja infekcije koriste se i molekularne metode koje daju precizne podatke o klonalnom poreklu bakterija izolovanih iz obolelih ljudi, namirnica i životinja. Međunarodnom trgovinom hrane isti tipovi bakterija mogu se pojaviti na geografski udaljenim lokacijama. Molekularna karakterizacija Salmonella je značajna zbog određivanja raznolikosti sojeva. Potrebno je da se izolati tipiziraju ne samo do nivoa vrste i serotipa nego i preciznije. Tipizacija je bitna za utvrđivanje epidemiološke povezanosti izolata, a genotipizacija podrazumeva direktnu analizu DNK. Geni rezistencije koji se od saprofita mogu preneti na patogene vrste imaju važnu ulogu u pojavljivanju rezistentnih i multirezistentnih sojeva. Međusobni odnos gena rezistencije i upotrebe antimikrobnih sredstava određuje jednačinu rezistencije na antimikrobna sredstva. Rezistencija predstavlja problem iako je salmoneloza samolimitirajuća infekcija zbog čega se terapija antibioticima primenjuje samo kod dece, starijih ljudi i u slučajevima sistemskih infekcija. Navedena problematika je aktuelna i u Crnoj Gori jer ne postoji stalan monitoring primene antimikrobnih sredstava kod domaćih životinja. Iz navedenih razloga cilj ove doktorske disertacije bilo je utvrđivanje prisustva Salmonella vrsta na farmama živine sa tri lokaliteta u Crnoj Gori, izolacija i identifikacija primenom standardnih mikrobioloških metoda, serotipizacija pomoću aglutinacije na predmetnom staklu (slide agglutination) uz primenu anti-O i anti-H seruma .(Staten Serum Institute, Danska). Primenom standardnih mikrobioloških metoda utvrđeno je prisustvo serovarijeteta Salmonella Enteritidis i Salmonella Tiphymurium. Serovarijeteti Salmonella .Gallinarum biotip Gallinarum i Salmonella Gallinarum biotip Pullorum nisu bile serološki tipizirane. Identifikacija navedenih serovarijeteta je potvrđena metodom multipleks PCR detekcijom amplifikovanih DNK fragmenata u agaroznom gelu.. Nakon digestije 50 izolata Salmonella enterica podvrste enterica odabranih prema lokalitetima i zastupljenosti pomoću SpeI restrikcionog enzma i njihovom analizom primenom PFGE utvrđeno je 5 različitih SpeI pulsotipova. Kod 10% ispitivanih sojeva ustanovljena je rezistencija na tetraciklin i streptomicin. Svi ispitivani serovarijeteti salmonela bili su osetljivi na amoksicilin sa klavulanskom kiselinom, enrofloksacin, ciprofloksacin, sulfametoksazol-trimetoprim, cefuroksim, ceftriakson i norfloksacin. Vrednosti MIK (minimalnih inhibitornih koncentracija) određenih antibiotika za odabrane sojeve serovarijeteta Salmonella Enteritidis primenom aparata VITEK 2 iznosile su za: piperacilin ≤ 4 – 64 μg/ml (S – R) , cefuroksim 4 – 32 μg/ml (S - R), cefuroksim aksetil 4 –32 μg/ml (S– R), cefiksim ≤ 0,25 – 2 μg/ml (S - I), ceftriakson ≤ 1 – 2 μg/ml (S - I) i minociklin ≤ 1 – 4 μg/ml (S), tetraciklin ≤ 1 μg/ml (S), tigeciklin ≤ 0,5 – 1 μg/ml (S), hloramfenikol ≤ 2 – 8 μg/ml (S), kolistin ≤ 0,5–1 μg/ml (S) i sulfametoksazol/trimetoprim ≤ 0,5 μg/ml (S). Salmonella is the most common cause of alimentary toxic infections among humans. They have been adapted to a number of warm-blooded animals. The infected animals do not exhibit symptoms and the treatment performs by finding of salmonella in routine health check. The secondary contamination by salmonella ois possible in during entire food chain. Apart from the routine microbiological analysis in detection of sources and pathways of spreading the infection, there are also used the molecular methods that provide accurate information about the clonal origin of bacteria isolated from diseased humans, food and animals. During international trade of food, the same types of bacteria can occur in geographically remote locations. Molecular characterization of Salmonella is important in determination of diversity of strains. It is necessary isolates to be typified not only to the level of species and serotypes but also more precisely. Typification is essential to determine the epidemiological connection of isolates. Genotyping includes a direct analysis of DNA. The resistance genes that can be transferred from saprophytes to pathogenic microorganisms play an important role in the emergence of resistant and multiresistant strains. The above mentoned is also a current issue in our country because there is a constant monitoring of using of antimicrobials drugs to farm animals. For these reasons, the aim of this dissertation is to examine the serovars of Salmonella in Montenegro, their molecular characterization using biomolecular methods based on isolation of DNA and subsequent amplification of serovar-specific genes.(multiplex PCR method and PFGE), and testing sensitivity, or resistance to antimicrobial drugs used in clinic vet practice.

U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije. Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima, radeno je metodom najverovatnijeg moguceg broja (MPN). Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a, utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pcela 110/kg. U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum. Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih metoda mikrobiologije. Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda. U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne rasporedenosti spora u uzorcima. In this study, the presence of Clostridium botulinum spores in different types of honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples was included in this investigation and samples originated from different regions of the Republic of Serbia. In addition to classical microbiological methods, after the dilution, enrichment, spinning and membrane filtration, PCR and multiplex PCR methods were used. Determination the number of spores in the PCR positive samples, was done using the most likely possible number (MPN) methodology. According to the results obtained by conventional microbiological methods and multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg. In one honey sample, spores of type B C. botulinum were detected, in one honey sample spores of type E, in two honey samples spores of types A and E, and in one sample spores of three types of C. botulinum (A, B, E) were detected. Type E Clostridium botulinum spores were detected in a sample of honey bees. In the same samples of honey or honey bees, using conventional qualitative and quantitative microbiological methods, spores of Clostridium botulinum were not detected. The detection of Clostridium botulinum spores with PCR, multiplex PCR and conventional microbiological methods in honey and honey bee samples is not possible without preenrichment. Conventional microbiological methods, including SRPS ISO 15213:2011, are not suitable for the detection of Clostridium botulinum spores in honey samples. Due to the low sensitivity of these methods, they can lead to false negative results. In the honey samples which served as a positive control and wich were contaminated with Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of one spore/g of honey. In order to detect Clostridium botulinum spores in honey and honey bees samples using PCR method, due to the small and/or unequal distribution of spores in the samples, the test had to be repeated at least three times for each sample.

In this study, the presence of Clostridium botulinum spores in different types of honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples was included in this investigation and samples originated from different regions of the Republic of Serbia. In addition to classical microbiological methods, after the dilution, enrichment, spinning and membrane filtration, PCR and multiplex PCR methods were used. Determination the number of spores in the PCR positive samples, was done using the most likely possible number (MPN) methodology. According to the results obtained by conventional microbiological methods and multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg. In one honey sample, spores of type B C. botulinum were detected, in one honey sample spores of type E, in two honey samples spores of types A and E, and in one sample spores of three types of C. botulinum (A, B, E) were detected. Type E Clostridium botulinum spores were detected in a sample of honey bees. In the same samples of honey or honey bees, using conventional qualitative and quantitative microbiological methods, spores of Clostridium botulinum were not detected. The detection of Clostridium botulinum spores with PCR, multiplex PCR and conventional microbiological methods in honey and honey bee samples is not possible without preenrichment. Conventional microbiological methods, including SRPS ISO 15213:2011, are not suitable for the detection of Clostridium botulinum spores in honey samples. Due to the low sensitivity of these methods, they can lead to false negative results. In the honey samples which served as a positive control and wich were contaminated with Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of one spore/g of honey. In order to detect Clostridium botulinum spores in honey and honey bees samples using PCR method, due to the small and/or unequal distribution of spores in the samples, the test had to be repeated at least three times for each sample. U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije. Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima, radeno je metodom najverovatnijeg moguceg broja (MPN). Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a, utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pcela 110/kg. U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum. Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih metoda mikrobiologije. Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda. U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne rasporedenosti spora u uzorcima.