Ispitivanje pouzdanosti seroloških, bakterioloških i molekularnih metoda u dijagnostici bruceloze pasa izazvane vrstom Brucella canis

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...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..

Examination of the reliability of serological, bacteriological and molecular methods in the diagnosis of canine brucellosis caused by Brucella canis

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..

Brucella canis at the territory of Serbia in the period from 2004. to 2011.

This study includes examination of dogs from the territory of Serbia during the period from 2004. To 2011. Most of the dogs were from the territory of Belgrade. The total of 193 blood serum samples of proprietary dogs and 120 blood serum samples of stray dogs were tested for the presence of antibodies against Brucella canis. For diagnostics there was used the method of slow serum agglutination in test tubes with 2- mercaptoethanol. During eight years’ period of investigation, out of 193 tested serums taken from proprietary dogs, 29 serum samples, or 15.03%, had undoubtedly positive titre of 1/200. During 2011. Out of 120 tested blood samples taken from stray dogs from Belgrade territory, positive titre of 1/200 had 8 samples, or 6.67%. The results of this investigation point out to a very high seroprevalence of antibodies against B.canis in dogs population from the teritiry of the Republic of Serbia. [Projekat Ministarstva nauke Republike Srbije, br. TR31088

Developmental Potential of Mouse Embryos without Extraembryonic Membranes in Modified Organ Culture

The long term stationary culture of postimplanatation embryos without extraembryonic membranes is a method to assess their developmental potential in vitro. The method was almost exclusively used on rat embryos, while mouse embryos were considered unsuitable due to their poor differentiation. In present study the postimplantation mouse embryos were used to verify potential of this method in mice. In addition, the course of in vitro differentiation was compared to embryo development in situ. Embryos were cultivated for maximum of 14 days and morphology and differentiation was analysed on serial semithin sections. Although anatomical relationships were lost from the beginning of the cultivation, the differentiation was only delayed, and the developmental potential after long term culture was comparable to those observed in rats. Therefore the advantages of long term cultivation could be utilized to analyse the differentiation of numerous lines of genetically modified mice with impaired postimplantation development

Detection and Phylogenetic Analysis of B2L Gene of ORF Virus from Clinical Cases of Sheep in Serbia

Infection of sheep by the ORF virus (ORFV) is very common in Serbia. ORFV is an economically important viral disease, distributed worldwide. Phylogenetic analysis based on the B2L gene of Serbian ORFV strains from two outbreaks that occurred in Serbia in 2016 is presented in this paper. Crust formation around the lips, nostrils, and udder was noted in all animals from the first outbreak, whilst in the second outbreak, all animals showed swollen and cyanotic lips and muzzle, with no visible crusts. Virus isolation was conducted using Vero cells. Cytopathic effects were evident on the third passage. However, all examined samples were positive using PCR. Phylogenetic analysis of the partial gene sequences (terminal gene regions were not included) encoding B2L gene of Serbian ORFV isolates showed 97.33-100.00% nucleotide and 92.86-100.00% amino acid similarity between each other. However, the viruses were divided into two clusters within the previously recognized Group 2, together with viruses from Croatia, Greece, Finland, China, South Korea and North America. This study is the first report of phylogenetic analysis of ORFV from Serbia and contributes to the data available in the GenBank database. The results of our investigation showed genetic diversity between ORFV strains in Serbia

Epizootiological service: One of the cornerstones of veterinary medicine

Факултет ветеринарске медицине представља један од факултета који пружају практична знања и вештине, а планови и програми појединих предмета дају солидну основу да по дипломирању лекар ветеринарске медицине зна да постави сумњу на неку заразну и паразитску болест, обави хируршку операцију, анализира резултате дијагностичких испитивања телесних флуида, уведе животињу у анестезију, обави телење, анализира резултате испитивања квалитета сточарских производа, апликује терапију у зависности од дијагнозе и потреба животиња, саветује сточара у вези технологије узгоја и биосиурносних мера итд. Кроз сваки од наведених (и ненаведених) послова, провлачи се један заједнички именитељ. Наиме, појединачни случајеви тј. пацијенти и најчешће позитиван исход третмана, не би значили пуно ако се не би анализовали са становишта правилности и учесталости појављивања болести и успеха спроведених мера. Као лекари ветеринарске медицине, често у помоћ позивамо статистичаре који нам са мање или више успеха, користећи статистичке методе, објасне како се појединачни случајеви болести уклапају у целу слику односа епизоотиолошких детерминанти: узрока (микроорганизма), пријемчиве врсте животиња (макроорганизма) и спољашњег фактора. Међутим, биолошки закони који представљају основу ветеринарске медицине често или нису до краја познати или по природи ствари нису елементи које статистичари уче. Отуда је од великог значаја да сваки лекар ветеринарске медицине буде и епизоотиолог и да на основу својих искустава (рада), покуша да пронађе закономерности поремећаја здравља, производних карактеристика и добробити у популацијама животињских врста као и да установи који су то фактори који утичу на појаву ових поремећаја, а са циљем изналажења мера за њихово уклањање и/или смањењe штета. Имајући у виду професионални профил епизоотиолога, може да се каже да епизоотиолошка служба обједињује податке о поремећајима здравља, производних карактеристика и добробити животиња и има задатак да анализује добијене информације, предлаже и спроводи мере за контролу, сузбијање и искорењивање пре свега заразних болести. Епизоотиолошка служба је у највећем броју случајева (земаља) организована и повезана како по вертикали тако и хоризонтално. Свакако, основа су ветеринари на терену који, поред власника и држаоца животиња, први постављају сумњу на присуство неког поремећаја здравља. Следећи ниво су ветеринарски инспектори и епизоотиолози који се налазе у оквиру специјализованих дијагностичких ветеринарских института. На врху епизоотиолошке пирамиде налази се оделење у оквиру Управе за ветерину које обједињује податке са терена али и тесно сарађује са осталим оделењима Управе. На тај начин, омогућава се да се епизиоотиолошка служба прикупља податке не само о заразним и паразитским болестима које често имају зоонотски потенцијал, већ и информације о поремећајима у продуктивности, подизању квалитативног и квантитативног стандарда производа животињског порекла и ризика по добробит животиња. Исто тако, епизоотиолошка служба у оквиру Управе за ветерину сарађује са Међународним Уредом за епизоотије (ОИЕ), поштујући принципе и критеријуме за размену података и међусобно извештавање Уреда и земаља чланица ОИЕ-а. Епизоотиолошка служба сарађује и са епидемиолошком службом у циљу контроле и сузбијања зооноза.The Faculty of Veterinary Medicine is one of the „hands on“ faculties whose curriculum offers a sound base for a graduate to diagnose an infective or parasitic disease, perform a surgical intervention, analyze results of body fluids tests, assist delivery, assess the quality of livestock products, apply treatment according to the diagnosis and need of the animal, offer council to the farmers, etc. In all the listed (and not listed) duties there is a common denominator, as individual cases would not be of relevance if not observed within the framework of disease regularity and incidence, as well as success of the performed measures. Doctors of veterinary medicine very often appeal to the help of statisticians which to a higher or lesser extent, with the aid of statistical methods, explain how individual cases fit within the whole picture of epizootiological factors: cause (microorganisms), susceptible animal (macro organism), and environmental factors. However, biological rules which represent the core of veterinary medicine very often are not elucidated, or are not taken into account by the statisticians. Thus, the importance for every veterinarian to find the epizootiologist within, and according to his/her knowledge and experience try to define the rules according to which a disease develops or production and welfare are affected. At the same time the veterinarian should be able to define which factors affect the presence of these disorders, all with the aim of prevention and/or damage mitigation. Bearing in mind the professional profile of an epizootiologist, it can be said that the epizootiological service encompasses data on health disorders, productive characteristics and animal welfare and within the Ministry of Agriculture has a role to analyze the obtained data, suggest and carry out control measures, combat and eradicate above all infectious diseases. Also, the Directorate for Veterinary Affairs forwards data to the International Office thus collaborating with the epidemiological service with the aim of zoonoses control and eradication

Examining the possibility of detecting brucella canis from tissue samples using bruce-ladder multiplex PCR assay

The goal of this study was to compare the results of serological and conventional bacteriological methods with the results obtained using multiplex PCR Bruce-ladder assay. Based on the obtained results, the usability of the assay was assessed in regard to rapid diagnosis of canine brucellosis directly from the samples of reproductive organs of infected dogs. Out of 225 blood samples, 33 (14.67%) had a positive agglutination reaction. In this study, out of the 225 assayed reproductive organs of dogs, B. canis was isolated from 3 samples (1.33%), while the PCR Bruce-ladder assay detected two positive samples (0.88%). Two dogs from which B. canis was isolated, an antibody titer of 1/200 was established in blood serums, and third dog from which B. canis was isolated was negative using the tube agglutination test. From a total of 225 assayed organ samples, a positive PCR reaction was obtained from two samples. The obtained results show that the tube agglutination method remains the first choice for the detection of dogs infected with B. canis. In addition, whenever possible, it is necessary to try isolation. It is desirable to attempt the detection of B. canis in tissues using PCR, but the results may not be treated as definitive and reliable

The performance of seven molecular methods for the detection of prrsv

Porcine Reproductive and Respiratory Syndrome is a viral disease of swine characterized by reproductive failure of breeding animals and respiratory disorders in all categories. The first PRRS case in Serbia was recorded in 2001 after illegal import of boar semen. PRRS is economically the most important disease due to significant direct and indirect losses. Today, for routine diagnosis of PRRS in infected herds serological methods (ELISA) and molecular methods are used. Although modern diagnostic techniques are very robust, exceptional diversity of the viral strains is often the obstacle for an accurate diagnosis. To estimate the performance of seven different methods for PRRSV genome detection, twenty samples were used. However, none of the methods was able to detect all PRRSV strains. The best sensitivity was obtained by combining two methods. Until today, there is no absolutely accurate test which enables the detection of all circulating strains

Presence of Mycoplasma bovis in bulk tank milk and associated risk factor analysis in Serbian dairy farms

Mycoplasma bovis (M. bovis) is a significant pathogen responsible for highly transmissible mastitis in cattle globally. It primarily spreads through colostrum, milk, and semen. Cows with persistent infections act as carriers, intermittently releasing the pathogen, making their milk a pivotal factor in infection transmission. Given the limited seroprevalence surveys in Serbia, this study aimed to detect M. bovis presence in bulk tank milk (BTM), determine route shedding, and evaluate infection risks. BTM samples were collected from 115 dairy farms across Serbia, with M. bovis DNA detected in 11 out of the 115 samples by real-time PCR. Additionally, M. bovis was detected in 1.30% of nasal swabs sampled from apparently healthy animals. A univariate analysis of the risk factors associated with M. bovis presence in the BTM samples revealed correlations with factors such as the breed, farm seropositivity, pre-milking and post-milking disinfection practices, farm type, cow population, milk yield, number of cows in the BTM samples, and parity. Seropositive farms exhibited the highest likelihood of M. bovis presence in milk. Moreover, pre- and post-milking disinfection practices and highly productive cows yielding over 8000 L of milk were identified as risk factors for PCR-positive BTM. In a multivariable mixed regression analysis, a risk factor for the presence of M. bovis infection in the BTM sample was the Holstein breed. These findings underscore a relatively high prevalence of M. bovis in BTM within Serbian dairy farms, suggesting a potential risk for M. bovis spreading through milk and oral route of calves’ infection

Molecular evidence of q fever agent coxiella burnetii in ixodid ticks collected from stray dogs in Belgrade (Serbia)

Q fever is a zoonotic disease caused by Coxiella burnetii, a gram-negative coccobacillus, which has been detected in a wide range of animal species, mostly domestic ruminants, but also in wild mammals, pets, birds, reptiles, arthropods (especially ticks), as well as in humans. Although the exposure to domestic animals in rural areas is regarded as the most common cause of the disease in humans, recent studies have shown that the role of pets in the epidemiology of Q fever has been increasingly growing. Although the primary route of infection is inhalation, it is presumed that among animals the infection circulates through ticks and that they are responsible for heterospecific transmission, as well as spatial dispersion among vertebrates. The aim of this study was to determine the presence and prevalence of C. burnetii in ticks removed from stray dogs, as well as to examine the distribution of tick species parasitizing dogs on the territory of Belgrade city. A PCR protocol targeting IS1111 repetitive transposon-like region of C. burnetii was used for the detection of C. burnetii DNA in ticks and the results were confirmed by sequence analysis. In total, 316 ticks were collected from 51 stray dogs - 40 females (78.43%) and 11 males (21.57%). Three species of ticks were identified: Rhipicephalus sanguineus (72.15%), Ixodes ricinus (27.53%) and Dermacentor reticulatus (0.32%). Out of 316 examined ticks, C. burnetii DNA was detected only in the brown dog tick R. sanguineus, with a total prevalence of 10.53% (24/228). The high prevalence of C. burnetii in R. sanguineus, which is primarily a dog tick, indicates the importance of dogs in the epidemiology of Q fever in the territory of Belgrade