Molecular detection and identification of microsporidia and viruses in honey bee colonies in Serbia

Primena molekularno – genetičkih metoda zauzima značajno mesto u dijagnostici oboljenja kod životinja izazvanih delovanjem različitih vrsta uzročnika. Ove metode, uglavnom zasnovane na lančanoj reakciji polimeraze (PCR), karakteriše zadovoljavajuća brzina izvođenja i visok stepen specifičnosti, odnosno osetljivosti. Mikrosporidije pčela, Nosema ceranae i Nosema apis, ispoljavanjem patogenog efekta na individualnom nivou, mogu dovesti do gubitka čitavih pčelinjih društava. Umnožavanjem mikrosporidijalne DNK, PCR tehnika omogućava istovremeno utvrđivanje prisustva i identifikaciju uzročnika u uzorku u kojem mikroskopskim pregledom nije utvrđeno prisustvo spora. Pojedine vrste virusa mogu biti uzročnici poremećaja zdravstvenog stanja pčela. Među najznačajnije viruse koji mogu naneti štete pčelinjim društvima ubrajaju se virus deformisanih krila (DWV), virus akutne paralize pčela (ABPV), virus mešinastog legla (SBV) i virus hronične paralize pčela (CBPV). Pomenuti virusi često izazivaju inaparentne infekcije koje, usled delovanja nespecifičnih faktora i drugih patogena, za kratko vreme mogu prerasti u klinički manifestne i dovesti do gubitka pčelinjih društava. Primenom tehnike Real-time RT-PCR bazirane na TaqMan probama, moguće je otkriti prisustvo virusne RNK koja se nalazi u niskoj koncentraciji u ispitanom uzorku. Ovo ima veliki značaj u dijagnostici virusnih infekcija kod pčelinjih društava koja ne ispoljavaju kliničke simptoma bolesti. Cilј ove doktorske disertacije je da se primenom molekularno-genetičkih metoda izvrši detekcija i identifikacija uzročnika četiri najčešće virusne infekcije (DWV, ABPV, SBV i CBPV) zastuplјene kod pčelinjih zajednica različite jačine koje potiču iz različitih krajeva Srbije...Molecular diagnostic in modern veterinary medicine plays an important role in diagnosis of animal health disorders. Major advantages of molecular diagnostic are quickness and high specificity and sensitivity in detection and identification of infectious and non-infectious agents. Microsporidia, Nosema ceranae and Nosema apis, as a consequence of pathogenic effects in individual bees may provoke losses of entire honey bee colonies. Amplification of microsporidial DNA using PCR allows simultaneous detection and identification of Nosema species in samples that appeared to be free from Nosema spores after microscopic examination.Honey bee viruses may have a significant impact on health status of honey bee colonies. Most common viruses considered to be able to cause damage to bee colonies include Deformed wing virus (DWV), Chronic bee paralysis virus (CBPV), Acute bee paralysis virus (ABPV) and Sacbrood virus (SBV). These viruses may exist in colonies in low infectious titres causing asymptomatic infection that due to the negative impact of non-specific factors and/or other pathogens may develop in to covert infection and lead to colony loss.High specificity and sensitivity of Real-time RT-PCR method based on TaqMan probes enables detection of very small amounts of viral RNA in bee samples originated from asymptomatic colonies which is advantage of this method compared to end point PCR. The aim of this work is detection and identification of four most common viruses (DWV, ABPV, SBV and CBPV) in honey bee colonies of different strength originated from different Serbian regions..

The concealed „gospel of greed“?: economy and its radical criticism

Рад излаже два типа критике економије, како као релативно младе научне дисциплине тако и као темељног и руководећег начела модерног капитализма изграђеног на прототипу похлепног homo economicusa. Оријентационо бисмо их могли назвати левом и десном или научном и религијском критиком, али се оне често сливају у једно инсистирајући на вредностима које надилазе ону једину вредност деификованог слободног тржишта и на неправедности коју оно, као организациони принцип друштва, намеће. У својим „конзервативним“ или „прогресивним“ варијантама, та кардинална критика се понекад закључује у оспоравању сваког надлештва економије над друштвом и сугестији потпуног одступања од владања према њеним узусима који, под алибијем доброћудног „властитог интереса“, славе грамзиво стицање, а понекад у „алтернативним“ визијама економисања које једнако не пристају на „логику капитала“. Закључује се да је, у сваком случају, реч о драгоценој проблематизацији коју, најзад, у савремености практикује и сама економија – само наизглед парадоксално – као одговорна наука афирмишући се тек довођењем у питање властитих принципа.The paper looks at two types of criticism of the economy, both as an emerging discipline, as well as an established and guiding principle of modern capitalism built on the prototypical greedy homo economicus. These two types of criticism could be defined as left and right, or scientific and religious critiques. However, they often merge into one view that insists on values that override the singular value of a self-defying free market, and the injustice that is imposed by that same market and which in turn serves as the organizational principle of the entire society. In its “conservative” or “progressive” variations, such cardinal criticism sometimes stems from bringing into question the dominance of economy over the entire society and moving away from the supremacy of those principles that, under the veil of benign self-interest, celebrate avaracious acquisition. Sometimes, such criticism could also emerge from “alternative” visions that similarly dismiss the „logic of capital“. It can be concluded that, in any case, such problematization is of great value and that, after all, it is practiced by the economy itself – though in a seemingly paradoxical manner – as a responsible science that can only be affirmed through bringing into question its founding principles

A molecular genetic approach to roebuck individual identification in the case of poaching in Serbia

Application of the molecular genetic methods in forensic cases dealing with wild animals has significantly increased recently. These techniques are practically used in order to help solving four key problems : determination of kind of the wild animal, geographic origin, kinship ties and individual identification. In this work the first case of introducing the examination of polimorphism of microsatelite genetic markers within forensic analysis in the cases of poaching in Serbia is presented. The objectives of this forensic analysis was to determine if the meat confiscated during house search of the suspect comes from roebuck origin (Capreolus capreolus), which remains had been found by a game warden in the field during closed season, where the suspect denied the offense, claiming that the meat comes from other roebuck that had been shot during the previous hunting season. DNK was isolated from the skin and fur samples taken from the roebuck corpse found in the woods, as well as from the frozen meat found in the suspect’s house. Both amplification and polimorphism examination of the eight microsatelite markers (ROE01, NVHRT21, NVHRT24, NVHRT48, NVHRT73, RT7 AND RT27) were carried out. In all the examined samples, the same pattern of variability of the tested microsatelites was determined, that is it was proved that DNK profiles of the samples taken from roebuck corpse were identical to DNK profile of the meat sample found in the suspect’s house. This result clearly indicates that all the examined biological samples originate from the same animal, and consequently represents forensically valid evidence in the case of roebuck poaching. [Projekat Ministarstva nauke Republike Srbije, br. III46002

Assessment of 17 microsatellite loci for their use in parentage verification and individual identification in the Balkan donkey breed

The aim of this study was to assess a panel of 17 microsatellites for parentage verification and individual identification in the endangered Balkan donkey breed. Allele frequencies for 17 microsatellite loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HTG4, HTG6, HTG7, HTG10, HMS1, HMS2, HMS3, HMS6, HMS7, LEX3 and VHL20) were determined in a 77 unrelated Balkan donkeys. Three loci (ASB2, HMS1 and ASB17) proved to be unsuitable and had been excluded from the investigation. Analysis of the remaining 14 loci revealed varied levels of polymorphism (three to 12 alleles), while the total number of observed alleles was 118 with an average of 8.42 per locus. Average values of observed heterozygosity and polymorphic information content (PIC) were 0.712 and 0.650, respectively. Twelve out of 14 microsatellite markers were highly informative with PIC values higher than 0.5. Only four loci were in HWE (HMS2, HMS6, HMS7 and HTG6). The obtained value of combined power of exclusion 0.9999) confirms usefulness of this microsatellite panel for parentage verification, while the value of combined power of discrimination of 0.9941 clearly approves the reliability of the panel for individual identification in Balkan donkeys

Retrospective analysis of the bluetongue outbreak in Serbia

Bluetongue, a vector-born disease caused by the Bluetongue virus (BTV) and transmitted by Culicoides biting midges, is considered to be one of the most important diseases of domestic ruminants. The first outbreak of bluetongue in Serbia was reported in 2001, when BTV serotype 9 was identified in sampled materials. In 2014, outbreak of BTV-4 in Serbia caused considerable economic losses affecting sheep, cattle and goats. During this outbreak, BTV-4 was recorded in 644 outbreaks within 49 municipalities, part of 17 administrative regions. From the total number of sheep kept in areas affected by bluetongue (n=1 748 110), 2 083 cases (0.2%) were proven to be BTV-4 infected. Total of 206 infected cattle and 24 infected goats were reported during this investigation period, which represents 0.06% and 0.03% of the total number of cattle and goats kept in affected areas, respectively. The highest incidence of infected sheep, cattle and goats was recorded on the territory covered by veterinary institute of Nis. Recorded lethality in cattle, sheep and goats was 18.45% (n=38), 48.10% (n=1002) and 54.17% (n=13), respectively. The peak of the outbreak was in September and October when 94.43% of the confirmed positive cases, regardless of the species, was recorded. Monitoring of bluetongue disease in Serbia relies on active surveillance programmes aimed at: (i) identification and tracing of susceptible and potentially infected animals and (ii) detection, distribution and prevalence of insect vectors. Vaccination of sheep is planned to be implemented as a control measure against bluetongue in Serbia

Validation of 10 microsatellite loci for their use in parentage verification and individual identification in the Yugoslavian Shepherd Dog Sharplanina

The aim of the study was to assess a commercially available microsatellite panel for use in paternity and identification analyses in the Yugoslavian Shepherd Dog Sharplanina, an ancient livestock guarding breed. Allele frequencies for 10 microsatellite loci (PEZ01, FHC2054, FHC2010, PEZ05, PEZ20, PEZ12, PEZ03, PEZ06, PEZ08 and FHC2079) were determined in 103 unrelated Yugoslavian Shepherd Dog individuals. The loci revealed varied levels of polymorphism (five to 12 alleles), with an average of 7.83 per locus. Average values of observed heterozygosity and polymorphic information content (PIC) were 0.64 and 0.66, respectively. Nine out of 10 microsatellite markers were highly informative with PIC values higher than 0.5. The obtained value of combined power of exclusion (0.9989) confirms usefulness of this panel of microsatellites for parentage verification, while the value of combined power of discrimination of 0.9999 clearly shows that the panel can conclusively identify individual dogs. In conclusion, the results obtained suggest that the selected set of commercially available microsatellite markers may be used as a routine tool for parentage verification and individual identification in the Yugoslavian Shepherd Dog. Although analysis of genetic variability of the Yugoslavian Shepherd Dog was not the primary focus of the study, the established values of major diversity indices disclose a highly variable gene pool in the breed

Morphological, biochemical and hematological characterization of endangered balkan donkey breed

The aim of the study was to establish morphometric, biochemical and hematological values for the endangered Balkan donkey breed (Serbia) and to explore the possible age dependence of the parameters tested. Inter-breed similarity of morphometric parameters was assessed by comparing the data obtained for the Balkan donkey with morphometric measurements of several previously characterized domestic donkey breeds. The study population included 74 donkeys, divided in two age groups (group A 3 years). In total, 18 morphometric, 13 hematological and 14 biochemical parameters were assessed. Significant morphometric differences (p < 0.05) in body length, head length, chest circumference and body weight were found between the two age groups. Significant differences in morphological parameters were revealed among the Balkan donkey and other donkey breeds (Catalonian, Croatian and Albanian), but results of cluster analysis demonstrated the smallest distance between the Balkan donkey and Albanian donkeys. The results of morphometric analyses showed consistency of the obtained values within the breed, and diversity as compared to other donkey breeds, and, thus, could be taken as referent for the Balkan donkey. Hematological and biochemical profiles obtained for the Balkan donkey were consistent with previous reports and within the recommended reference ranges. White blood cell, mid cell and granulocyte counts, showed significantly higher (p < 0.05) values in donkeys under 3 years of age, while the only biochemical parameter affected by age was alkaline phosphatase. The information gained through characterization of the Balkan donkey breed provides a basis for conservation and development of the breed standard

Komparacija metoda za detekciju mikrosporidia iz roda Nosema kod medonosne pčele (Apis mellifera)

Two microsporidia species of the Nosema genus cause nosemosis in the adult honeybee: N. apis and N. ceranae. For diagnostic purposes and the determination of infection level various microscopic and molecular biological methods are used. The aim of this research was to compare the reliability of the traditional microscopic assessment and two PCR techniques: simplex- and duplex-PCR. Honey bee samples were taken from 150 colonies. Microscopic examination, performed according to the recommendations of the OIE, revealed Nosema spores in 68.67% samples analysed, whilst with the simplex-PCR method all samples (100.0%) proved positive. On the other hand, duplex-PCR method used for the identification of Nosema species resulted in 84.0% positive samples, all of which were N. ceranae. Our recommendation of the simplex-PCR method for the monitoring of honey bees in field conditions is based on its higher reliability than the microscopic assessment in the detection of low-level infections, as well as its potential for the detection of vegetative Nosema sp. stages; thus the early detection and timely prevention of Nosema infection would be possible. Nosema species identification is simplest and most cost-effective if performed with the duplex-PCR analysis. However, the simplex-PCR is more reliable, thus, it is suggested that samples that were negative when assessed with microscopy and duplex-PCR analysis undergo simplex-PCR.Dve vrste mikrosporidija roda Nosema uzročnici su nozemoze kod odrasle medonosne pčele: N. apis i N. ceranae. Za postavljanje dijagnoze i utvrđivanje stepena infekcije koristi se nekoliko mikroskopskih i molekularno-bioloških metoda. Cilj našeg rada bilo je poređenje pouzdanosti tradicionalne mikroskopske metode i dve PCR metode: simplex- i duplex-PCR. Pregledano je ukupno 150 uzoraka pčela. Mikroskopskim pregledom, obavljenim prema preporukama OIE, prisustvo spora Nosema utvrđeno je u 68,7% uzoraka. Međutim, simplex-PCR metodom dobijeni su pozitivni rezultati u svih 150 uzorka (100,0%). Sa druge strane, primenom duplex-PCR metode infekcija je ustanovljena kod 84,0 %; u svim slučajevima determinisana je vrsta N. ceranae. Veća pouzdanost simplex-PCR metode u odnosu na mikroskopski pregled, kako u otkrivanju infekcije malog intenziteta, kao i mogućnost detekcije vegetativnih oblika nozeme, navodi nas da preporučimo uvođenje simplex-PCR metode kao obavezne za praćenje stanja pčelinjih društava na terenu; time bi se postigla rano utvrđivanje prisustva infekcije i blagovremena prevencija njenog širenja. Specijska identifikacija mikrosporidija roda Nosema najjednostavnija je i najisplativija metodom duplex-PCR. Međutim, simplex-PCR ima veću pouzdanost, te preporučujemo da se uzorci koji su negativni na osnovu mikroskopskog pregleda i duplex-PCR analize ispitaju i simplex-PCR metodom

Effects of chestnut tannins supplementation of prepartum moderate yielding dairy cows on metabolic health, antioxidant and colostrum indices

This study investigated the effects of dietary supplementation with chestnut tannins (CNT) on metabolic and antioxidant status of prepartum cows along with their colostrum quality. Pregnant multiparous Holstein cows were paired according to parity and body condition score, and assigned either to a diet supplemented with 20 g/d of commercially available product containing chestnut tannins (CNT, n=20) or to an unsupplemented control diet (CON, n=20) for the last 25±2 d of pregnancy. Serum metabolite, insulin and antioxidant capacity indices were measured in blood samples taken at d 25 and d 5 before expected parturition. Chemical composition and IgG concentration were determined in colostrum samples collected from the first milking postpartum. The addition of CNT led to lower BUN (P=0.02) and consequently higher serum glucose (P=0.02) and insulin (P<0.01) concentrations which were associated with lower circulating NEFA (P<0.01) and BHBA (P<0.01) in CNT group than those of CON. The serum paraoxonase 1 (PON 1) activity and total antioxidant capacity (T-AOC) were higher at-5 d in CNT than in CON (P<0.01, P=0.03; respectively). Close-up CNT improved lactose percentage and IgG concentration (P=0.03, P=0.04; respectively) and tended to improve percentage of protein and SNF (Solid Not Fat) in primary colostrum (P=0.06, respectively), without affecting colostrum fat and total solid (P=0.98, P=0.43; respectively). Supplementation of CNT in the diet during close-up period did not have adverse effects on metabolic profiles prepartum. Instead, this feeding regimen was more beneficial to antioxidant capacity and colostrum quality than feeding the control diet

Evidence of West Nile virus (WNV) circulation in wild birds and WNV RNA negativity in mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016

West Nile virus (WNV) is a zoonotic flavivirus whose transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. Bridge vectors can transmit WNV to mammal species potentially causing West Nile Fever. Wild bird migration is a mode of WNV introduction into new areas. The Danube Delta Biosphere Reserve (DDBR) is a major stopover of wild birds migrating between Europe and Africa. The aim of this study was to investigate the presence of WNV in the DDBR during the 2016 transmission season in wild birds and mosquitoes. Blood from 68 wild birds (nine different species) trapped at four different locations was analyzed by competitive ELISA and Virus Neutralization Test (VNT), revealing positive results in 8/68 (11.8%) of the wild birds by ELISA of which six samples (three from juvenile birds) were confirmed seropositive by VNT. Mosquitoes (n = 6523, 5 genera) were trapped with CDC Mini Light traps at two locations and in one location resting mosquitoes were caught. The presence of WNV RNA was tested in 134 pools by reverse transcription quantitative PCR (RT-qPCR). None of the pools was positive for WNV-specific RNA. Based on the obtained results, WNV was circulating in the DDBR during 2016