Raccoon dog rabies surveillance and post-vaccination monitoring in Lithuania 2006 to 2010

<p>Abstract</p> <p>Background</p> <p>Oral rabies vaccination (ORV) in rabies infected regions should target the primary rabies vector species, which in Lithuania includes raccoon dogs as well as red foxes. Specific investigations on ORV in raccoon dogs are needed e.g. evaluation of vaccine effectiveness under field conditions. The objective of the current study was to investigate the efficacy of the ORV programme 2006-2010 in Lithuania by examining the number of rabies cases and estimating the prevalences of a tetracycline biomarker (TTC) and rabies virus antibodies in raccoon dogs.</p> <p>Methods</p> <p>From 2006 to 2010, 12.5 million rabies vaccine-baits were distributed by aircraft. Baiting occurred twice per year (spring and autumn), targeting raccoon dogs and red foxes in a 63,000 km²area of Lithuania. The mandibles of raccoon dogs found dead or killed in the vaccination area were analyzed by fluorescence microscopy for the presence of the TTC. Rabies virus sera neutralizing anti-glycoprotein antibody titres were determined using an indirect ELISA method and seroconversion (> 0.5 EU/ml) rates were estimated.</p> <p>Results</p> <p>During the study period, 51.5% of raccoon dog mandibles were positive for TTC. 1688 of 3260 tested adults and 69 of 175 tested cubs were TTC positive. Forty-seven percent of raccoon dog serum samples were positive for rabies virus antibodies. 302 of 621 investigated adults and 33 of 95 investigated cubs were seropositive. In the same time 302 of 684 and 43 of 124 tested samples were TTC and ELISA positive in spring; whereas 1455 of 2751 and 292 of 592 tested samples were TTC and ELISA positive in autumn. There was a positive correlation between the number of TTC and antibody positive animals for both adult and cub groups.</p> <p>Conclusions</p> <p>ORV was effective in reducing the prevalence of rabies in the raccoon dog population in Lithuania. The prevalence of rabies cases in raccoon dogs in Lithuania decreased from 60.7% in 2006-2007 to 6.5% in 2009-2010.</p

Filogenetsko istraživanje djelomičnih sekvencija gena G terenskih izolata virusa bjesnoće u Litvi.

The objective of this study was to evaluate the molecular epidemiology of rabies virus G-gene partial sequences in rabies positive field samples, isolated in different regions of Lithuania, and to compare the sequences phylogenetically with various rabies virus isolates of wild carnivores from the Baltic Sea region, Europe and Russia. Twenty brain samples of rabid animals, collected during the period 2006-2011, were investigated. Multiple alignments of 358 nt G gene (nt 3324-3682) sequences were performed using ClustalW with default settings. Phylogenetic and molecular evolutionary analyses were conducted using the Neighbor-Joining method (NJ) in MEGA version 4. The phylogenetic investigations of rabies virus glycoprotein indicate that the Lithuanian rabies virus isolates’ G-sequences were closely related and showed 89.8-98.9 % of nt identity. The G-sequences between Lithuanian raccoon dog and red fox rabies virus isolates were more conservative (97.2 % nt identity) than the rabies virus isolates inside the raccoon dog group (95.1 % nt identity). Two Lithuanian raccoon dog rabies virus isolate G-sequences were rather divergent (90.8 and 91.2 % nt identity) and were closely associated with rabies virus isolate G-sequences from Estonia (92.5 % nt identity), Russia (91.0 %) and Poland (89.3 %) respectively. The comparative investigation of G-sequences between the Lithuanian rabies virus isolates and different isolates from Germany, Slovenia and France indicated 84.2 % nt identity, whereas the G-sequences of Lithuanian rabies virus isolates and rabies virus isolate G-sequences from Russia were 88.1 % nt identical.Svrha ovog rada bila je odrediti molekularnu epidemiologiju djelomičnih sekvencija gena G virusa bjesnoće u terenskim uzorcima pozitivnima na bjesnoću iz različitih područja Litve te ih filogenetski usporediti s različitim izolatima virusa bjesnoće iz divljih mesojeda na području Baltičkog jezera, Europe i Rusije. Pretraženo je 20 uzoraka tkiva mozga bijesnih životinja prikupljenih u razdoblju od 2006. do 2011. Višestruka poravnanja sekvencija od 358 nukleotida gena G (nt 3324-3682) učinjena su pomoću programa ClustalW. Filogenetska i molekularno evolucijska analiza provedena je metodom združivanja genetski najsličnijih sojeva (engl. Neighbour-joining method) u MEGA verziji 4. Filogenetska istraživanja glikoproteina virusa naznačuju da su G-sekvencije izolata litavskoga virusa bjesnoće usko srodne i pokazuju 89,8-98,9 % nt identičnosti. G-sekvencije izolata virusa bjesnoće iz litavskog rakunskog psa i crvenih lisica bile su sačuvanije (97,2 % nt identičnosti) nego izolati virusa bjesnoće izdvojeni iz rakunskog psa (95,1 % nt identičnosti). G-sekvencije dvaju izolata iz litavskih rakunskih pasa bile su prilično divergentne (90,8 i 91,2 % nt identičnosti) i usko srodne s G-sekvencijama izolata virusa bjesnoće iz Estonije (92,5 % nt identičnosti), Rusije (91,0 %) i Poljske (89,3 %). Komparativno istraživanje G-sekvencije litavskih izolata virusa bjesnoće i različitih izolata iz Njemačke, Slovenije i Francuske pokazalo je 84,2 % nt identičnosti, dok je identičnost između G-sekvencije između litavskih izolata virusa bjesnoće i ruskih izolata bila na razini 88,1 % nt identičnosti

Cross-Sectional Study on the Prevalence and Factors Influencing Occurrence of Tick-Borne Encephalitis in Horses in Lithuania

Various animal species have been evaluated in depth for their potential as Tick-borne encephalitis virus (TBEV) sentinel species, although evidence for equine capacity is incomplete. Therefore, a comprehensive cross-sectional stratified serosurvey and PCR analysis of selected horses (n = 301) were performed in TBEV endemic localities in Lithuania. Attached and moving ticks (n = 241) have been collected from aforementioned hosts to evaluate natural infectivity of TBEV vectors (Ixodes spp.) in the recreational environments surrounding equestrian centers. All samples were screened for TBEV IgG and positive samples were confirmed by virus neutralization test (VNT). 113 (37.5%) horses from all counties of Lithuania tested positive for TBEV IgG, revealing age and sex indifferent results of equine seroprevalence that were significantly dependent on pedigree: horses of mixed breed were more susceptible to infection possibly due to their management practices. TBEV prevalence in equine species corresponded to TBEV-confirmed human cases in the precedent year. As much as 3.9% of horses were viraemic with TBEV-RNA with subsequent confirmation of TBEV European subtype. 4/38 of tested tick pools were positive for TBEV-RNA (Minimal infectious rate 1.2%). Several unknown microfoci were revealed during the study indicating areas of extreme risk close to popular human entertainment sites. The study provides important evidence in favor of horses’ usage as sentinel species, as equines could provide more detailed epidemiological mapping of TBEV, as well as more efficient collection of ticks for surveillance studies

SEROSURVEY OF WILD BOAR POPULATION FOR PORCINE PARVOVIRUS AND OTHER SELECTED INFECTIOUS DISEASES IN LITHUANIA

Abstract Wild boar (Sus scrofa scrofa) population in Lithuania was tested for antibodies against porcine parvovirus (PPV), foot and mouth disease virus (FMDV), Brucella sp., classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), and Aujeszky&apos;s disease virus (ADV). Blood serum samples were collected from shot wild boars during hunting season in 2004. Using ELISA no antibodies against FMDV (n=504), Brucella sp. (n=23), CSFV (n=591), SVDV (n=12), and ADV (n=24) were detected. With respect to PPV haemagglutination inhibition test was used. Altogether 254 serum samples were tested. It was found that 67 (26.4%) wild boars were seronegative, 64 (25.2%) had PPV antibody titres from 1 to 5 log2 (2-32, possibly passive maternal immunity), 12 (4.7%) -from 6 to 7 log2 (64-128, possibly declining passive maternal immunity or rising active postinfective immunity), 30 (11.8%) -from 8 to 10 log2 (256-1 024, active postinfective immunity), 75 (29.5%) -from 11 to 13 log2 (2 048-8 192, high active postinfective immunity) and in 6 (2.4%) boars the titre was 14 log2 (16 384, very high active postinfective immunity). Thus, our data showed that PPV circulates actively in Lithuanian wild boar population

Phylogenetic Analysis of the Rabies Virus N-coding Region in Lithuanian Rabies Isolates

Rabies infection among wild and domestic animals constitutes a well-known problem in Lithuania, but only one dog rabies virus isolate sequence (1992) from Lithuania was used in the European rabies virus phylogenetic analysis. The objective of this work was to determine nucleoprotein (N) gene sequences and genetically characterize the rabies virus isolates in order to learn which virus group (biotype) is circulating in reservoir species in Lithuania. Classical rabies virus isolate nucleoprotein (N) gene sequences from different parts of Lithuania were found to be closely related to each other and demonstrated nucleotide identity from 97.7 to 100% and could be placed in one lineage with 100% bootstrap support. All 12 sequences of raccoon dogs, red foxes, dogs and marten rabies viruses exhibited 97.7 - 99.0% identity to previously published sequences from Eastern parts of Poland, Estonia, Finland, and the North-Eastern part of Russia. Phylogenetic analysis revealed that all Lithuanian strains belong to the North East Europe (NEE) group of rabies virus

E. coli paplitimas mažmeninės prekybos realizuojamose kepenėlėse, jų atsparumas antimikrobinėms medžiagoms

Tyrimų tikslas – ištirti E. coli paplitimą mažmeninės prekybos realizuojamose vištų kepenėlėse, kaip viename iš populiariausių vištienos subproduktų, ir nustatyti šių bakterijų atsparumą antimikrobinėms medžiagoms. Iš skirtingų mažmeninės prekybos vietų surinkti įvairių Lietuvos gamintojų šviežių žalių vištų kepenėlių mėginiai (240 mėginių) ir atlikti E. coli bakteriologiniai tyrimai. Iš mėginių išskirtas vienas šimtas E. coli padermių (41,7 proc.) ir nustatytas jų atsparumas antimikrobinėms medžiagioms. Tyrimams taikytas mikroskiedimų metodas plokštelėse, naudojant 14 skirtingų antimikrobinių medžiagų. Rezultatai vertinti pagal ribines jautrumo reikšmes, nurodytas Europos antimikrobinio jautrumo tyrimų komiteto (EUCAST) duomenų bazėje. Nustatyta, jog dažniausiai tirtosios E. coli padermės pasižymėjo atsparumu streptomicinui (100 proc.), ampicilinui (60 proc.), nalidikso rūgščiai (50 proc.), ciprofloksacinui (47 proc.) ir tetraciklinui (45 proc.). Atsparių amikacinui padermių nenustatyta. Nustatytas retas atsparumas cefoksitinui (2 proc.), ceftiofurui (7 proc.), chloramfenikoliui (10 proc.), amoksicilino ir klavulano rūgšties kombinacijai (15 proc.). Mažiausios slopinamosios koncentracijos, didesnės nei plokštelėse esančios antimikrobinių medžiagų koncentracijos, nustatytos visoms tirtoms medžiagoms, išskyrus amikaciną. Rasta daug E. coli padermių, kurioms inaktyvuoti reikėjo didelio MSK ampicilino, nalidikso rūgšties, sulfonamidų ir tepraciklino kiekio. Tyrimų duomenys rodo potencialią riziką vartotojams (užsikrėsti atspariomis bakterijomis ar jų perduodamais atsparumo veiksniais per maisto gamybos grandinę), nes šios antimikrobinės medžiagos naudojamos tiek žmonių, tiek ir veterinarinėje medicinojeThe objective of this study was to estimate the prevalence and the antimicrobial resistance of E. coli that contaminates raw chicken liver as one of the most popular poultry sub-product sold in retail markets. Two hundred and forty samples of fresh raw chicken liver were obtained from national poultry producers in different retail marketing sites and tested for the presence of E. coli. One hundred E. coli strains (41.7%) were isolated and tested for antimicrobial susceptibility. The MICs of 14 antimicrobial agents were determined for each of the isolates using the broth microdilution method with custom-made microtitre plates. EUCAST cut-off values were used for the interpretation of susceptibility of isolated bacteria to antimicrobial agents. The most frequent resistances were demonstrated to streptomycin (100 %), ampicillin (60%), nalidixic acid (50%), ciprofloxacin (47%) and tetracycline (45%). No resistant strains were found to amikacin. Law percentage of resistant strains was recorded to cefoxitin (2%), ceftiofur (7%), chloramphenicol (10%) and amoxicillin/clavulanic acid (15%). MIC’s values above dilution ranges were found to all antimicrobials except amikacin. The highest numbers of resistant strains that demonstrated resistance to the highest concentrations of antimicrobial agents were found to ampicillin, nalidixic acid, sulphonamides and tetracycline. The data demonstrate potential risk during food preparation for consumers in the context of resistant E. coli as abovementioned antimicrobial agents are used in veterinary and human medicine as wellLietuvos sveikatos mokslų universitetasMatematikos ir statistikos katedraVytauto Didžiojo universiteta

Prevalence and phylogenetic analysis of hepatitis E virus in pigs, wild boars, roe deer, red deer and moose in Lithuania

Abstract Background Hepatitis E virus (HEV) is one of the major causes of acute viral hepatitis worldwide. In Europe, food-borne zoonotic transmission of HEV genotype 3 has been associated with domestic pigs and wild boar. Controversial data are available on the circulation of the virus in animals that are used for human consumption, and to date, no gold standard has yet been defined for the diagnosis of HEV-associated hepatitis. To investigate the current HEV infection status in Lithuanian pigs and wild ungulates, the presence of viral RNA was analyzed by nested reverse transcription polymerase chain reaction (RT-nPCR) in randomly selected samples, and the viral RNA was subsequently genotyped. Results In total, 32.98 and 22.55% of the domestic pig samples were HEV-positive using RT-nPCR targeting the ORF1 and ORF2 fragments, respectively. Among ungulates, 25.94% of the wild boar samples, 22.58% of the roe deer samples, 6.67% of the red deer samples and 7.69% of the moose samples were positive for HEV RNA using primers targeting the ORF1 fragment. Using primers targeting the ORF2 fragment of the HEV genome, viral RNA was only detected in 17.03% of the wild boar samples and 12.90% of the roe deer samples. Phylogenetic analysis based on a 348-nucleotide-long region of the HEV ORF2 showed that all obtained sequences detected in Lithuanian domestic pigs and wildlife belonged to genotype 3. In this study, the sequences identified from pigs, wild boars and roe deer clustered within the 3i subtype reference sequences from the GenBank database. The sequences obtained from pig farms located in two different counties of Lithuania were of the HEV 3f subtype. The wild boar sequences clustered within subtypes 3i and 3h, clearly indicating that wild boars can harbor additional subtypes of HEV. For the first time, the ORF2 nucleotide sequences obtained from roe deer proved that HEV subtype 3i can be found in a novel host. Conclusion The results of the viral prevalence and phylogenetic analyses clearly demonstrated viral infection in Lithuanian pigs and wild ungulates, thus highlighting a significant concern for zoonotic virus transmission through both the food chain and direct contact with animals. Unexpected HEV genotype 3 subtype diversity in Lithuania and neighboring countries revealed that further studies are necessary to understand the mode of HEV transmission between animals and humans in the Baltic States region

Otpornost na antibiotike bakterija Enterococcus spp. izdvojenih iz stoke u Litvi.

105 isolates of Enterococcus spp. were selected as representative samples from different herds of pigs (n=25), cattle (n=46) and poultry (n=34). Antimicrobial susceptibility was determined according to epidemiological cut-off values. The highest number of strains demonstrated epidemiological resistance to lincomycin (78%), tetracycline, (65%), flavomycin (59%) and erythromycin (55%). The smallest number of strains was resistant to linezolid (1%) and tigecycline (2%). Six percent of all tested strains were epidemiologically resistant to ciprofloxacin, vancomycin and daptomycin. Cattle isolates showed the most frequent resistance to fl avomycin (71%), lincomycin (54%), tetracycline (45%), streptomycin (40%) and erythromycin (40%). Enterococci isolated from pigs showed the highest resistance to tetracycline and lincomycin (92%), erythromycin (76%), kanamycin (56%) and streptomycin (52%). All strains isolated from poultry were epidemiologically resistant to lincomycin. The most frequent resistance of poultry strains was also demonstrated to tetracycline (72%), erythromycin (63%), streptomycin (50%), flavomycin (48%) and tylosin (47%). However all strains isolated from poultry were susceptible to chloramphenicol, quinupristin/dalfopristin, linezolid and bacitracin.Ukupno je 105 izolata bakterija Enterococcus spp. bilo odabrano kao predstavnici iz različitih uzgoja svinja (n = 25), goveda (n = 46) i peradi (n = 34). Osjetljivost prema antimikrobnim tvarima određivana je na osnovi epidemioloških graničnih vrijednosti. Većina izolata bila je otporna prema linkomicinu (78%), tetraciklinu (65%), flavomicinu (59%) i eritromicinu (55%). Najmanje izolata bilo je otporno prema linezolidu (1%) i tigeciklinu (2%). Šest posto svih pretraženih sojeva bilo je otporno na ciprofloksacin, vankomicin i daptomicin. Izolati iz goveda bili su najčešće otporni na flavomicin (71%), linkomicin (54%), tetraciklin (45%), streptomicin (40%) i eritromicin (40%). Enterokoki izdvojeni iz svinja bili su najotporniji prema tetraciklinu i linkomicinu (92%), eritromicinu (76%), kanamicinu (56%) i streptomicinu (52%). Svi izolati iz peradi bili su otporni prema linkomicinu. Sojevi iz peradi također su bili najčešće otporni prema tetraciklinu (72%), eritromicinu (63%), streptomicinu (50%), flavomicinu (48%) i tilozinu (47%). Međutim svi sojevi izdvojeni iz peradi bili su osjetljivi prema kloramfenikolu, kvinupristinu/dalfopristinu, linezolidu i bacitracinu