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Background: Hantaviruses are etiological agents of emerging zoonotic diseases worldwide, including hemorrhagic fever with renal syndrome (HFRS). A number of hantavirus species is known to be present in Europe. In Serbia, existing data on hantavirus presence and prevalence rely in serological findings. In this study, molecular analysis was performed in order to characterize HFRS causing hantaviruses in Serbia. Methods: Sixty four serum samples of HFRS cases, previously found seropositive to anti-hantaviral anti-bodies, were included in the study. Partial hantaviral L and S segments were PCR amplified producing 390nt and 598nt amplicons, respectively, in parallel with human beta-actin mRNA as external reverse transcription positive control. Hantavirus specific PCR products were DNA sequenced in both direction and the obtained sequences phylogenetically confirmed and analyzed. Results: PCR detection of hantavirus L and S genome segments was positive in 18/64 and 11/64 tested samples, respectively. Positive PCR results involved samples obtained from different locations, mostly from central and southern parts of Serbia. All the obtained sequences were identified as Dobrava-Belgrade virus (DOBV). In the phylogenetic analysis sequences from Serbia tended to cluster in distinctive, geographically related clusters. Conclusions: Our findings indicate DOBV as the main HFRS causing hantavirus in Serbia, the site of its initial isolation. (C) 2019 Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The disease caused by the West Nile virus (WNV) has been known since 1937 when it was described for the first time in Uganda. After spreading to Europe and the Middle East, the disease has changed its primary location. Today WN infection is a significant health problem in the world. As a result of the current epizootiological and epidemiological situation in Europe studies on the occurrence of WND were introduced in Serbia, also. The investigation on the presence of WNV antibodies was intensified in the period from 2008 to 2012. In this period a total of 3618 serum samples were tested from 18 localities (2736 animal sera from 8 different species and 882 human sera samples). The method applied was gel immunodiffusion and the representative samples were confirmed by the plaque reduction test (PRNT-90). Out of the total number of samples WNV antibodies were present in 3.97% horses, 0.93% dogs, 0.31% poultry and 1.36% man. In one horse serum sample there was a positive reaction with a positive control serum, thus indicating the presence of WNV antigen. The results have confirmed that WNV antibodies are present in 9 out of 18 tested locations in the Republic of Serbia. The percentage of seropositive samples varies from 0.42% in Pozarevac (horses and humans) up to 6.45% in Novi Pazar (dogs). Out of the investigated species the highest seropositivity was recorded in horses (3.97%), and lowest in poultry (0.31%). WNV is present and widespread in the Republic of Serbia, thus enabling distribution mapping. Bolest koju izaziva virus Zapadnog Nila (WNV) je poznata još od 1937. kada je po prvi put opisana u Ugandi. Posle širenja virusa u Evropu i na Bliski istok, bolest je promenila prvobitnu lokaciju. Danas je WN infekcija značajan zdravstveni problem u svetu. Kao rezultat trenutne epizootiološko-epidemiološke situacije u Evropi, uvedena su istraživanja prisustva WNV i u Srbiji. Istraživanja prisustva antitela za WNV intenzivirana su u periodu od 2008. do 2012. godine. U ovom periodu ukupno je ispitano 3618 seruma sa 18 lokaliteta (2736 seruma životinja, od 8 različitih vrsta i 882 seruma ljudi). Korišćen je metod imunodifuzije u gelu a reprezentativni uzorak potvrđen je testom neutralizacije plaka (PRNT- 90). Od ukupnog broja uzoraka na prisustvo antitela za WNV bilo je pozitivno 3,97% konja, 0,93% pasa, 0,31% domaće živine i 1,36% ljudi. U serumu jednog konja pojavila se pozitivna reakcija sa pozitivnim kontrolnim serumom, ukazujući na prisustvo antigena WNV. Dobijeni rezultati su potvrdili da su antitela za WNV prisutna u 9 od 18 ispitivanih lokacija u Republici Srbiji. Procenat seropozitivnih varira od 0.42% u Požarevcu (konji i ljudi) do 6,45% u Novom Pazaru (psi). Od ispitivanih vrsta najveća seropozitivnost je registrovana kod konja (3,97%), a najniža kod živine (0,31%). Raspoloživi podaci ukazuju da je WNV prisutan i značajno raširen u Republici Srbiji, što je omogućilo i mapiranje njegovog širenja.

The disease caused by Influenza viruses has been well known for a very long time. In the recent period there has been noted an occurrence of pandemics caused by Influenza viruses type A with a high rate of mortality. The ongoing pandemic caused by avian influenza virus serotype H9N9 began in Hong Kong in 1992, and another pandemic caused by serotype H5N1 began in China (Hong Kong) in 1999. The world wide spreading of these viruses occurred due to migratory birds. Avian influenza was confirmed in Serbia in 2007. The goal of this study was to examine whether the avian influenza viruses type A circulate in the region of the Obedska bara marsh, which is a famous resort for many birds in Serbia, as well as many birds migrating from Europe to Africa and vice versa. The samples of blood sera of many animal species (123 samples from fowl, 64 samples from donkeys, 40 samples from horses) were tested by serologic reaction of inhibition of haemmaglutination (IHA) for the presence of antibodies to influenza A subtypes H5N1, H5N2, H5N3, H7N1 and H7N2. Also, the samples of blood sera of experimental chicken exposed to wild life in Obedska bara (sentinel species) were tested. Antibodies to subtypes H5N1, H5N2, H5N3, H7N1 and H7N2 were found in chicken from Dec, Boljevci, Petrovcic and Kupinovo villages but no antibodies were found in blood sera from hams from Dobanovci, Jakovo, Becmen and Surcin villages. From 23 samples from ducks antibodies were detected in 3 samples, and from 22 geese blood sera antibodies were found in 4 samples. From a total of 40 horse blood sera tested one was tested positive, and from 64 donkey sera 17 were positive for the presence of antibodies for avian influenza type A. In blood sera of experimental chicken antibodies were found by subtype H5N1 with corrections with H5N2 and H7N1. Bolest izazvana influenca virusima poznata je od davnina. U novije vreme, javljale su se pandemije izazvane influenca A-virusima sa visokom stopom smrtnosti. Sadašnja pandemija avijarne influence pojavila se u Hongkongu 1992. godine sa serotipom H9N9, a sa H5N1 počela je 1997. godine u Kini (Hongkong). Globalizaciju ove pandemije učinile su migracione ptice. Pojava bolesti registrovana je i u našoj zemlji 2007. godine. Cilj rada je bio da se ispita prisustvo virusa influence ptica tipa A kod životinja na lokalitetu Obedska bara, poznatom rezervatu brojnih ptica u Srbiji, kao i odmorištu ptica koje migriraju iz Evrope u Afriku i obrnuto. Reakcijom inhibicije hemaglutinacije (HI test) ispitivani su uzorci krvnih seruma raznih životinja (123 uzorka pernate živine, 64 seruma magaraca i 40 seruma konja) na prisustvo antitela za influencu A - podtipovi H5N1, H5N2, H5N3, H7N1 i H7N2. Takođe su ispitivani i uzorci seruma eksperimentalnih pilića izloženih kontaktu sa divljom prirodom (sentinel jedinke) na području Obedske bare. Specifična antitela za podtipove H5N1, H5N2, H5N3, H7N1 i H7N2 utvrđena su kod pilića u selima Deč, Boljevci, Petrovčić i Kupinovo, dok u serumima kokošaka poreklom iz Dobanovaca, Jakova, Bečmena i Surčina antitela nisu nađena. Od 23 uzorka krvnog seruma pataka, specifična antitela su utvrđena kod 3 uzorka i u 4 od 22 uzorka seruma gusaka. Od 40 ispitivanih uzoraka krvnih seruma konja antitela su ustanovljena u jednom uzorku, dok je u uzorcima seruma magaraca prisustvo specifičnih antitela utvrđeno u 17 od 64 ispitivana uzorka. U serumima eksperimentalnih pilića antitela su nađena za podtip virusa influence H5N1 sa koreakcijama sa antigenima podtipova H5N2 i H7N1. PR Projekat Ministarstva nauke Republike Srbije, br. TP 21047.

We assayed the presence of antibodies specific for Rickettsia typhi, R. akari, and R. conorii in sera of persons from several localities in Serbia with different geographic, climatic, and lifestyle characteristics. Sera from 140 patients with unclear clinical symptoms and 273 healthy persons were tested for the presence of rickettsiae-specific antibodies by indirect immunofluorescence assay. In this study, for the first time we detected the presence of rickettsiae from the spotted fever group in Serbia. We detected the presence of antibodies against R. conorii in the samples from all tested localities. The proportion of positive cases was low in the plain agricultural areas but reached up to 23% in the mountain areas. We also observed a significant number of cases positive for antibodies against R. akari. Antibodies specific for the antigens of R. typhi were detected in only 2 samples from the municipality of Pec (Kosovo region). These findings contribute to the prevalence of Rickettsia species in Southeast Europe. Our study also revealed a dramatic lack of awareness of rickettsioses among medical personnel and pointed to the need for urgent measures that would help improve the current situation in the region.

SUMMARYHantaviruses are endemic in the Balkans, particularly in Serbia, where sporadic cases and/or outbreaks of hantaviral human disease have been reported repeatedly, and evidenced serologically. Here, we present genetic detection of Dobrava-Belgrade virus (DOBV) hantaviral sequences in wild rodents trapped in central Serbia. All the animals were pre-screened serologically by indirect immunofluorescence (IF) test and only those with a positive finding of hantaviral antigens were further tested by polymerase chain reaction. Of the total of 104 trapped animals, 20 were found to be IF positive and of those three were positive for hantaviral RNA: one Microtus arvalis for Tula virus, and one each of Apodemus agrarius and Glis glis for DOBV. Phylogenetic analysis of the obtained sequences implies putative DOBV spillover infection of A. agrarius and G. glis from Apodemus flavicollis. However, future investigations should help to identify the most common natural host and geographical distribution of DOBV in its reservoir hosts in Serbia.

Introduction Ehrlichiosis is a bacterial zoonosis transmitted by hematophagous arthropods - ticks. In humans, it occurs as monocytic, granulocytic, and ewingii ehrlichiosis. Pathological process is based on parasitic presence of Ehrlichia organisms within peripheral blood cells - monocytes and granulocytes. Case Outline Fifty-two year old patient was admitted to hospital due to high fever of over 40°C that lasted two days, accompanied with chills, muscle aches, malaise, loss of appetite, headache, confusion, breathing difficulties, and mild dry cough. The history suggested tick bite that occurred seven days before the onset of disease. Doxycycline was introduced and administered for 14 days, causing the disease to subside. Indirect immunofluorescence assay was used to analyze three serum samples obtained from this patient for Ehrlichia chaffeensis antibodies, and peripheral blood smear was evaluated for the presence of Ehrlichia and Ehrlichia aggregation into morulae. Conclusion Ehrlichiosis should be considered in each case where there is a history of tick bite together with the clinical picture (high fever, chills, muscle aches, headache, generalized weakness and malaise, and possible maculopapular rash). The presence of Ehrlichia chaffeensis antibodies was confirmed in a patient with the history of tick bite, appropriate clinical picture and indirect immunofluorescence assay. This confirmed the presence of human monocytotropic ehrlichiosis, a disease that is uncommonly identified in our country. Uvod Erlihioza je bakterijska zoonoza koja se prenosi hematofagnim artropodama - krpeljima. Kod ljudi se javlja kao monocitna, granulocitna i ewingii erlihioza. Patološki proces je posledica unutar ćelijskog parazitiranja erlihije u monocitima i granulocitima periferne krvi. Prikaz bolesnika Bolesnik star 52 godine primljen je na odeljenje zbog visoke dvodnevne febrilnosti (preko 40°C) koja je praćena drhtavicom, bolovima u mišićima, malaksalošću, gubitkom apetita, glavoboljom, konfuznošću, otežanim disanjem i oskudnim suvim kašljem. U anamnezi je dobijen podatak o ujedu krpelja sedam dana pre prijema. Laboratorijski nalazi su ukazali na trombocitopeniju, leukopeniju, anemiju i povećanje aktivnosti transaminaza u serumu. Bolesnik je lečen doksiciklinom 14 dana, nakon čega su se tegobe povukle. Metodom indirektne imunofluorescencije analizirana su tri uzorka seruma ovog bolesnika na prisustvo antitela na bakteriju Ehrlichia chaffeensis i pregledan je uzorak razmaza periferne krvi na prisustvo erlihija i konglomerata erlihije u morule, koje predstavljaju citoplazmatske vakuole. Zaključak Kod bolesnika s podatkom o ubodu krpelja, odgovarajućom kliničkom slikom i serološkim testom indirektne imunofluorescencije dokazana su antitela za bakteriju Ehrlichia chaffeensis, što ukazuje na humanu monocitotropnu erlihiozu, bolest koja se kod nas retko dokazuje. Na erlihiozu treba misliti kada uz kliničku sliku (visoka febrilnost, groznica, bolovi u mišićima, glavobolja, opšta slabost i malaksalost, eventualno makulopapulozna ospa) postoji podatak o ubodu krpelja.

Five hundred and forty-four rodents and small mammals were trapped in various regions of Yugoslavia and examined. Antihantaviruses, immunofluorescent (IF) antibodies were detected in the blood samples of 129 animals. Antigens were detected in the lung samples of 139 animals. Sixty-seven animals tested positive for both the presence of antibodies in the sera and antigens in the lungs. Hantaviral antibodies and/or antigens were detected most in the yellow-necked mouse (Apodemus flavicollis) (88/189), the wood mouse (Apodemus sylvaticus (28/146), the striped field mouse (Apodemus agrarius) (10/64), house mouse (Mus musculus) (14/29), and the Norway rat (Rattus norvegicus) (14/21). Five other species of rodents and insectivora were infrequently infected. Hantavirus infection in small mammals, according to age, gender, location and species indicated a disparity from region to region prior to and during the epidemic. RA 1; HFRS; Ecology; Human Disease; BD.

Five hundred and forty four rodents and small mammals were trapped in various regions of Yugoslavia and examined. Antihantaviruses, immunofluorescent (IF) antibodies were detected in the blood samples of 129 animals. Antigens were detected in the lung samples of 139 animals. Sixty-seven animals tested positive for both the presence of antibodies in the sera and antigens in the lungs. Studies on the immune status of healthy people in various HFRS endemic areas were conducted. Blood samples from over 700 forest workers, farmers, and other individuals with considerable outdoor exposure were collected and tested serologically for antibodies to hantaviruses. Approximately 336 individuals possessed hantaviral antibodies. In 1989, an HFRS epidemic occurred throughout Yugoslavia, over 600 individuals were hospitalized and 15 deaths occurred. The epidemic occurred in all six republics and two provinces of Yugoslavia, in both previously recognized and newly recognized foci areas.