Molekularni dokaz i karakterizacija velogenog soja virusa newcastleske bolesti u čvorka u Makedoniji.

The objective of this study was to assess virulence using molecular methods and to phylogenetically characterize Newcastle disease virus (NDV) detected in common starlings (Sturnus vulgaris) in Macedonia. Nucleotide sequencing of the cleavage site of the F gene revealed an amino acid pattern specific for virulent strains of NDV with phenylalanine at position 117 and multiple basic amino acids between positions 112 and 116, i.e. 112RRQKR*FIG119. The 374 bp region of the fusion (F) gene used for phylogenetic analyses revealed that the detected strain belongs to subgenotype VIId of the class II NDV, and it is similar to virulent viruses detected in back-yard poultry in Macedonia in 2005 and 2006, as well as to viruses from poultry and wild birds detected in Serbia and Bulgaria in 2006 and 2007. Epidemiological data suggest that the common starlings were probably infected as a result of an ongoing epizootic in domestic poultry and they did not have a role in the primary introduction and spread of the virus to domestic poultry. This study represents the first report of the detection and molecular and phylogenetic characterization of virulent NDV from wild birds in Macedonia.Cilj ovog istraživanja bio je molekularnim metodama odrediti virulenciju i filogenetska obilježja virusa newcastleske bolesti (NB) dokazanog u čvorka (Sturnus vulgaris) u Makedoniji. Pokazalo se da nukleotidni slijed na mjestu cijepanja gena F daje aminokiselinski sastav specifičan za virulentne sojeve virusa NB s fenilalaninom na poziciji 117 i uzastopnim bazičnim aminokiselinama između pozicija 112 i 116, tj. 112RRQKR*FIG119. Filogenetska analiza područja od 374 bp fuzijskog (F) gena pokazala je da izdvojeni soj pripada podtipu VIId skupine II virusa NB, a srodan je virulentnim sojevima dokazanima u domaće peradi u Makedoniji 2005. i 2006. kao i s virusima izdvojenima iz peradi i divljih ptica u Srbiji i Bugarskoj 2006. i 2007. Epizootiološki podatci upućuju na zaključak da su čvorci vjerojatno bili zaraženi kao posljedica pojave NB u domaće peradi te nisu imali nikakvu ulogu na pojavu i širenje virusa na domaću perad. Ovo je prvo izvješće o dokazu, molekularnim i filogenetskim obilježjima virulentnog virusa NB u divljih ptica u Makedoniji

Bruceloza u trudnoći: prikazi slučaja s različitim ishodima u endemskom području

Different outcomes of brucellosis in pregnancy regarding the fetus/neonate and the mother are described. Medical records of five pregnant women with brucellosis were retrospectively analyzed. Patients were treated in several departments of infectious diseases in the Republic of Macedonia between 1995 and 2009. The diagnosis of brucellosis was based on clinical findings compatible with the disease supported by detection of specific antibodies. Pregnancy outcomes in patients were as follows: spontaneous abortion, intrauterine fetal death, premature delivery in two cases (one with twin pregnancy) and term delivery. One of the women experienced relapse. Follow-up results of neonates showed no infection and their normal growth and development. Brucellosis, especially if acquired in early pregnancy, can have an impact on pregnancy outcome. In endemic regions, in pregnant women with persisting fever and unspecific manifestations one should always have in mind brucellosis. In these areas, cases with unexplained spontaneous abortion, intrauterine fetal death and premature delivery should also be investigated for brucellosis.Opisuju se različiti ishodi bruceloze u trudnoći u odnosu na fetus/novorođenče i majku. Retrospektivno su analizirani medicinski zapisi za pet trudnica s brucelozom. Bolesnice su liječene u nekoliko klinika za zarazne bolesti u Republici Makedoniji u razdoblju od 1995. do 2009. godine. Dijagnoza bruceloze temeljena je na kliničkim nalazima sukladnima s bolešću i potkrijepljena otkrivanjem specifičnih protutijela. Ishodi trudnoće u ovih bolesnica bili su sljedeći: spontani pobačaj, intrauterina smrt fetusa, prijevremeni porođaj u dva slučaja (jedan s blizanačkom trudnoćom) i terminski porođaj. Recidiv je nastupio kod jedne od ovih žena. Rezultati praćenja novorođenčadi pokazali su odsutnost infekcije te normalan rast i razvoj. Bruceloza, osobito ako je stečena u ranoj trudnoći, može utjecati na ishod trudnoće. U endemskim područjima brucelozu treba uvijek imati na umu kod trudnica s ustrajnom groznicom i nespecifičnim manifestacijama. U ovim područjima testiranje na brucelozu treba provoditi i u slučaju neobjašnjenog spontanog pobačaja, intrauterine smrti fetusa i prijevremenog porođaja

Molekularna detekcija ehrlichia canis u populaciji pasa -kućnih ljubimaca u Severnoj Makedoniji

Canine monocytic ehrlichiosis (CME) is a widespread, tick-borne, canine disease, caused by an obligate intracellular bacterium, Ehrlichia canis. The main vector, a brown-dog tick, Rhipicephalus sanguineus, is widely distributed, especially in areas with tropic, subtropic, or Mediterranean climates (Central and South America, Eastern and Western Asia, Africa, Australia and Southern Europe). The study performed in 2012, by Stefanovska et al., determined a seroprevalence of 18.7% of E. canis among the Macedonian dog population. Up to date, the presence of E. canis, using molecular diagnostic methods, has not been investigated in Macedonia. Therefore, this study aimed to confirm the presence of E. canis, in the pet-dog population on the territory of the city of Skopje, North Macedonia, using a highly sensitive multiplex Real-Time PCR method (qPCR). Whole blood samples from 80 dogs of different breeds and ages, with clinical symptoms of CME and positive serology result for the presence of antibodies against E.canis, were collected for analyses. Out of 80 dogs, 36 (45%) were found as positive. The present work reports the first molecular detection of E. canis in pet dogs on the territory of the city of Skopje, Macedonia.Monocitna erlihioza pasa (CME) je široko rasprostranjena bolest pasa, koja se prenosi krpeljima, a uzrokuje je obligatno-intracelularna bakterija Ehrlichia canis. Glavni vektor, smeđi krpelj pasa, Rhipicephalus sanguineus, široko je rasprostranjen, posebno u oblastima sa tropskom, subtropskom ili mediteranskom klimom (Centralna i Južna Amerika, Istočna i Zapadna Azija, Afrika, Australija i Južna Evropa). U Studiji izvedenoj 2012. godine, Stefanovska i saradnici su utvrdili da među severnomakedonskom populacijom pasa, seroprevalencija E. canis iznosi 18,7%. Do danas, prisustvo E. canis, koristeć i molekularne dijagnostičke metode, nije istraženo u Severnoj Makedoniji. Stoga je ova studija imala za cilj da potvrdi prisustvo E. canis u populaciji kuć nih ljubimaca na teritoriji grada Skoplja u Severnoj Makedoniji, koristeć i visoko osetljivu multiplex Real-Time PCR (qPCR). Za analize su prikupljeni uzorci pune krvi od 80 pasa različitih rasa i uzrasta, sa kliničkim simptomima CME i pozitivnim serološkim rezultatom na prisustvo antitela protiv E.canis. Od 80 pasa, 36 (45%) je ocenjeno kao pozitivno. Ovaj rad izveštava o prvom molekularnom otkrivanju E. canis kod pasa kuć nih ljubimaca na teritoriji grada Skoplja, Severna Makedonija

Evaluation of west nile virus diagnostic capacities in veterinary laboratories of the mediterranean and black sea regions

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities

Classical and molecular characterization of pigeon paramyxovirus type 1 (PPMV-1) isolated from backyard poultry – first report in Macedonia

Aim of this study was to characterize pigeon variant of Newcastle disease virus (NDV) isolated from backyard poultry using classical and molecular methods. In standard hemagglutination inhibition (HI) test both polyclonal NDV antiserum and monoclonal antibodies 161/617 specific for pigeon variants of NDV showed inhibition of heamagglutination of the isolated virus. Intracerebral pathogenicity index (ICPI) has shown that the isolate is mesogenic virus (ICPI = 0.81). One-step RT-qPCR for detection of M gene was performed indicating a presence of NDV and RT-qPCR for discrimination between lentogenic and velogenic strains based on F gene was also performed indicating a presence of virulent NDV. A portion of the F gene was amplified and sequenced for determination of virulence and phylogenetic characterization. The F protein cleavage site sequence of the isolate had multiple basic amino acids at residues 112–116 and a phenyl alanine at residue 117 (112RRQKR*F117) which is typical for velogenic strains. The nucleotide sequence of 374 bp was aligned to begin at nt 47 and finish at 420 immediately after the cleavage site and compared with other reference strains from the region and worldwide. In the phylogenetic tree, the isolate clustered into genotype VIb, typical for PPMV-1. This strain is phylogenetically very similar to other PPMV-1 isolated from pigeons in Macedonia. Poultry infected with PPMV-1 can spread the virus in the absence of clinical signs, thus PPMV-1’s are constant threat to domestic poultry. This is the first report of evidenced spillover of PPMV-1 into poultry in Macedonia

Phenotypic and genotypic characteristics of enterocin producing enterococci against pathogenic bacteria

The study investigated the antimicrobial activity of 13 enterococcal strains (E. faecalis -8, E. faecium-2, E. hirae-2, E. spp.-1) isolated from our traditional cheeses against pathogen microorganisms. Also, it includes the detection of the following enterocin structural genes: enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin Q, enterocin EJ97 and cytolysin by using PCR method. All isolates inhibited growth of L. monocytogenes and L.innocua. One isolate had a broader antimicrobial activity. None of the isolates showed inhibitory activity against S. enteritidis, E. coli and Y. enterocolitica. The genes enterocin P, cytolysin and enterocin A were the most frequently detected structural genes among the PCR positive strains. No amplification was obtained in two strains E. faecalis-25 and E. faecalis-86. Three different genes were identified in some strains. With the exclusion of strains possessing a virulence factor, such as cytolysin, producers of more than one enterocins could be of a great technological potential as protective cultures in the cheese industry

Gaps in Brucellosis Eradication Campaign in Sheep and Goats in Republic of Macedonia: Lessons Learned

Aim To identify why “test and slaughter policy” for eradication of brucellosis did not significantly reduce the prevalence in sheep and goats in Macedonia. Method Coverage of sampled vs expected number of sheep and goats, absolute number of positive animals, prevalence, frequency distribution, and classes of disease prevalence were retrospectively analyzed at the village level for 2004-2006. A comparative analysis of the disease prevalence in the investigated villages was also performed. The percentage of slaughtered animals was analyzed for 2000-2006. Results We found differences between the expected and actual number of sampled animals, which were related to the type of livestock breeding. Traditionally maintained flocks and migratory flocks were considered to be responsible for the transmission of the disease. The absolute number of positive animals and the number of infected vs non-infected holdings did not decrease over the study period. Most of the villages had between 1 and 10 positive animals. Between 2000 and 2006, 55% of the positive animals were slaughtered, 41% in 2001 and up to 79% in 2002. Moreover, in 2005 and 2006, 34% and 53% of sheep and goats were found to be positive at the slaughter line, respectively, demonstrating that only 21%-23% of the infected animals were correctly removed from the herds. Conclusion Based on the findings of this study, Macedonia changed its control strategy from “test and slaughter” to a vaccination policy for sheep and goats in 2008

Serological diagnostic of maedi-visna (MVV) in sheep and caprine arthritis encephalitis virus (CAEV) in goats in Macedonia

Maedi-Visna in sheep and CAEV in goats are diseases caused by lentiviruses with very similar antigenic characteristics. This diseases are very important for the animal husbandry because of the economical losses they cause (reduced milk yield, lower body weight, deaths, therapy expenses). The results in this article, represents the first serological confirmation for the existence of this diseases in Macedonia. Investigations showed high percent of seroprevalence in the examined herds. The seroprevalence ranges from 60.3% for Maedi-Visna to 55.8% for CAEV. Despite the presence of clinical signs of the disease, the mortality is low, witch is related to the presence of the causal agent in the field i.e. the endemic character of this diseases

Surveillance of avian influenca viruses in farmed poultry in 2009 in Republic of Macedonia

The aim of the study was to determine the presence and distribution or to confirm the absence of avian influenca viruses in farmed poultry in the poultry production systems 1 and 2 from the eight statistical regions in R. Macedonia. Total number of 1215 cloacal swabs from poultry were sampled. Each sample was processed and analysed by both molecular (RRT-PCR) and classical virology methods (virus isolation and identification). All samples gave negative result for presence of avian influenza viruses. Commercial poultry production systems have biosecurity measures preventing the entry of pathogens i.e avian influenza viruses, therefore resulting with no circulation of these viruses in the sampled farmed poultry flocks