Veterinary RecordVolume 172, Issue 1 p. 22-23 Letter Clinical rabies in cattle imported into Croatia Ivana Lojkić, Corresponding Author Ivana Lojkić [email protected] Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, Croatiae-mail: [email protected]Search for more papers by this authorTomislav Bedeković, Tomislav Bedeković Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this authorŽeljko Čač, Željko Čač Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this authorNina Lemo, Nina Lemo Department of VirologySearch for more papers by this authorŽeljko Cvetnić, Željko Cvetnić Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this author Ivana Lojkić, Corresponding Author Ivana Lojkić [email protected] Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, Croatiae-mail: [email protected]Search for more papers by this authorTomislav Bedeković, Tomislav Bedeković Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this authorŽeljko Čač, Željko Čač Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this authorNina Lemo, Nina Lemo Department of VirologySearch for more papers by this authorŽeljko Cvetnić, Željko Cvetnić Department of Immunology, Croatian Veterinary Institute, Savska Cesta 143, Zagreb, CroatiaSearch for more papers by this author First published: 05 January 2013 https://doi.org/10.1136/vr.e8619Citations: 5Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1Bourhy H. Kissi B. Audry L. Smreczak M. Sadkowska-Todys M. Kulonen K. Tordo N. Zmudzinski J. F. Holmes E. C. (1999) Ecology and evolution of rabies virus in Europe. Journal of General Virology 80, 2545 –2557 2Dean D. J. Abelseth M. K. Athanasiu P. (1996) The fluorescence antibody test. In Laboratory Techniques In Rabies. Eds F. X. Meslin M. M. Kaplan H. Koprowski. World Health Organization. ppp 88–93 3Dietzgen R. G. Calisher C. H. Kurath G. Kuzmin I. V. Rodriguez L. L. Stone D. M. Tesh R. B. Tordo N. Walker P. J. Wetzel T. Whitfield A. E. (2011) Family Rhabdoviridae. In Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. Eds A. M. Q. King M. J. Adams E. B. Carstens E. J. Lefkowitz. Elsevier Academic Press. pp 686 –714 4Heaton P. R. Johnstone P. Mcelhinney L. M. Cowley R. O'Sullivan E. Whitby J. E. (1997) Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses. Journal of Clinical Microbiology 35, 2762 –2766 5Hudson L. C. Weinstock D. Jordan T. Bold-Fletcher N. O. (1996) Clinical features of experimentally induced rabies in cattle and sheep. Journal of Veterinary Medicine 43, 85 –95 6Huelsenbeck J. P. Ronquist F. (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754 –755 7Johnson N. Fooks A. R. Valtchovski R. MüLler T. (2007) Evidence for trans-border movement of rabies by wildlife reservoirs between countries in the Balkan Peninsular. Veterinary Microbiology 120, 71 –76 8Lojkić I. Čač Ž Bedeković T. Lemo N. Brstilo M. Mülier T. Freuling C. M. (2012) Diversity of currently circulating rabies virus strains in Croatia. Berliner und Münchener Tierärztliche Wochenschrift 125, 249 –254 9Mcelhinney L. M. Freuling C. M. Cragg W. Stankov S. Lalosević D. Lalosević V. Mülier T. Fooks A. R. (2011) Molecular diversity and evolutionary history of rabies virus strains circulating in the Balkans. Journal of General Virology 92, 2171 –2180 10Picard-Meyer E. Robardet E. Moroz D. Trotsenko Z. Drozhzhe Z. Biarnais M. Solodchuk V., Others (2012) Molecular epidemiology of rabies in Ukraine. Archives of Virology 157, 1689 –1698 11Turcitu M. Barboi G. Vuta V. Mihai I. Boncea D. Dumitrescu F. Codreanu M. Johnson N. Fooks A. R. MüLler T. Freuling C. M. (2010) Molecular epidemiology of rabies virus in Romania provides evidence for a high degree of heterogeneity and virus diversity. Virus Research 150, 28 –33 Citing Literature Volume172, Issue1January 2013Pages 22-23 ReferencesRelatedInformation

The aim of this study was to perform a diagnosis and molecular characterisation of bovine leukosis virus (BLV) on a Croatian dairy farm. For that purpose we compared the diagnostic tools used in our study, made sequence and phylogenetic analysis of our BLV and tried to describe the diffi culties in the process of EBL eradication on the examined farm after reintegration into Croatian territory after the war. From 1998 to 2008 blood samples from a dairy farm in the North-eastern part of Croatia were tested serologically using AGID and ELISA. In 2002, 2003 and 2004 37%, 22% and 10% of animals were serologically positive, respectively. After the initial eradication steps, the disease reappeared in 2008, when all examined blood samples reacted positively in BLV-specifi c nested PCR. Finally, at the end of 2010, after an extended eradication program, which included the implementation of PCR together with regular ELISA testing for detection of positive animals, the farm obtained the status of “free of BLV”. Sequence and phylogenetic analysis of 524-nt fragment of BLV env gp51 encoding gene showed the greatest identity with the Croatian BLV genotype 8 isolates, so it was phylogenetically clustered with those isolates.

To eliminate cytotoxic effects of colostrum on cells, a modified virus neutralization test (VNT) for the detection of Bovine viral diarrhea virus-specific neutralizing antibodies in colostrum was developed. The new test was compared to the World Organization for Animal Health-recommended VNT and the results evaluated. The agreement of the new test compared to the standard VNT was determined to be 98%, whereas sensitivity and specificity of the modified VNT compared to the standard VNT were 100%. Bovine viral diarrhea virus-specific antibodies were detected in 42 sera samples and 38 colostrum samples. The antibody titers in serum and colostrum showed a high correlation (n = 56, r = 0.9719, P < 0.001). The modified virus neutralization technique described herein succeeds in eliminating cytotoxic effects and can be readily applied for the detection of specific antibodies against other infectious agents in colostrum.

The virus neutralisation test is used for the quantitation of specific antibodies in serum samples. However, the success of the test depends on the quality of samples. In the case of poor quality samples, a cytotoxic effect can be observed and the results of the test can be compromised. Additionally, the cytotoxic effect limits the use of different substances, such as muscle extract or liquid from thoracic cavity (thoracic liquid), as a sample for the detection of rabies virus neutralising antibodies in the follow-up of fox oral vaccination campaigns. To eliminate the cytotoxic effect, a modified fluorescent antibody virus neutralisation (mFAVN) test was developed and evaluated. In the mFAVN test, inocula were removed after a 1 h and the cytotoxic effect was prevented. According to the results obtained, the specificity of the mFAVN test compared to the FAVN test was 88.8% and the sensitivity was 94.4%. The diagnostic validity of the test was 0.99 (CI = 0.98-1.00). To evaluate the possibility of using muscle extract and thoracic liquid as samples for the virus neutralisation test, 102 sera, muscle extract and thoracic liquid samples of dog origin were tested with the mFAVN test. The correlation between sera and muscle extracts was 87.9% (r = 0.88, p < 0.001). The correlation between sera and thoracic liquid was 94.2% (r = 0.94, p < 0.001). These findings indicated that both muscle extract and thoracic liquid could be used as samples for detection of rabies virus neutralising antibodies in the follow-up of oral vaccination campaigns. To evaluate the level of elimination of the cytotoxic effect, the 102 samples of sera, muscle extracts and thoracic liquid of dog origin were also tested in parallel using the mFAVN and FAVN tests. In the mFAVN test, no instance of cytotoxic effect was observed in the cells. In the FAVN test, two sera (1.9%), 35 muscle extracts (34.3%) and 56 thoracic liquid samples (54.9%) showed cytotoxic effect. The results of this study strongly suggest that cytotoxic effect can be eliminated completely from the rabies virus neutralising antibody detection tests used in the follow-up of oral vaccination campaigns and that very poor quality samples, such as muscle extract and thoracic liquid, can be used. (C) 2013 Elsevier B.V. All rights reserved.

The aim of this study was to estimate the influence of category, herd size, common grazing and management as risk factors in maintaining bovine viral diarrhoea infection in dairy herds. A total of 987 sera samples obtained from 202 heifers, 653 cows and 132 calves from 103 herds in Croatia were examined by enzyme-linked immunosorbent assay. In order to establish the prevalence of persistently infected cattle, 35 herds were selected. Ear notch tissue samples from all animals in selected herds (n = 2284) were collected and analyzed by antigen enzyme-linked immunosorbent assay. The true prevalence of specific antibodies was 61.61% and the estimated prevalence of exposure to bovine viral diarrhoea virus at the herd level was 100%. The prevalence of persistently infected animals was 0.53% and the prevalence of persistently infected herds was 20%. The antibodies prevalence was higher in cows, in herds that use common pasture and in larger herds (P < 0.001). The prevalence of persistently infected animals was not connected with the herd size but production management on big farms contributed to maintaining the virus. The obtained results suggest that production management was an important risk factor in bovine viral diarrohea epidemiology. High prevalence of antibodies and high prevalence of persistently infected herds requires implementation of control and eradication programs at a national or even regional level. The presented data complete the BVD epidemiological investigations from this part of Europe.

Sylvatic rabies has been present in Croatia for more than three decades, with the red fox (Vulpes vulpes) as the main reservoir. The present epidemic of sylvatic rabies in Croatia started already in 1977 and in the past ten years the disease has become enzootic in the entire country and thus represents a considerable veterinary and public health threat. A genetic characterization and phylogenetic analysis of rabies virus isolates (RABV) from Croatia was performed using panel of 32 selected rabies-positive brain samples from domestic and wild animals collected between 2008 and 2010. Based on the comparison of 367-nucleotide sequences of a conserved region of the nucleoprotein (N) gene (nucleotides 75-441), the phylogenetic analysis revealed a low genetic diversity of currently circulating RABV strains in Croatia. 18 RABV isolates mainly originating from Eastern Croatia clustered with the formerly established Eastern European (EE) lineage, and the rest (14) were identical with the West European (WE) group. Both phylogenetic groups seem to coincide in central regions on both sides along the Save River. A high sequence identity in the N gene of the RABV isolates from neighbouring countries was found.

Between 2007 and 2011, a total of 1937 sera samples and five spleen samples from nine Croatian dairy herds were tested for the bovine viral diarrhea virus (BVDV) using virus isolation and the immunoperoxidase test. BVDV was detected in 13 persistently infected (PI) cattle with a non-cytopathogenic biotype, while in five animals with fatal mucosal disease, isolates from spleen samples were of the cytopathogenic biotype. To reveal the genetic typing of Croatian BVDV isolates, viral RNA was extracted from infected cell cultures and amplified by RT-PCR, with primers targeting the 5'-UTR and the Npro gene, followed by direct sequencing of purified PCR products. Sequence and phylogenetic analysis of the 5'-UTR genome region determined that all Croatian isolates belonged to BVDV genotype 1; 11 isolates were grouped with BVDV-lb and 7 with BVDV-If viruses. The phylogenetic tree inferred by the Bayesian approach, using combined 5'-UTR/Npro, supported clustering of Croatian isolates in two subgroups. The deduced aminoacid sequence of the Npro region revealed 5 sites unique for four domestic BVDV-If isolates.

To investigate the degree of genetic variability of bovine leukemia virus (BLV) strains circulating in Croatia, 29 isolates from the six largest dairy farms were examined by PCR for a segment of the gp51 env gene, followed by DNA sequencing and phylogenetic analysis. The nucleotide sequences were compared with other previously characterized BLV strains from different geographical areas, comprising all seven known BLV genotypes. The Croatian sequences showed six to eight nucleotide substitutions: six silent substitutions and two amino acid changes. Four of those substitutions were within epitopes. In comparison to the sequences of other BLV genotypes, our isolates showed the closest relationship to genotype 1 isolates PL-3252 (FJ808585) and AL-148 (FJ808573) from Argentina. The degree of variation between our sequences and those of genotype 1 was 0.2- 4.6 %. In phylogenetic trees based on 400-nt and 519-nt sequences, all of the Croatian sequences clustered separately from the other sequences, revealing a new genotype.

BEDEKOVIC, T., N. LEMO, I. LOJKIC, Ž. CVETNIC, Ž. CAC, J. MADIC: Bovine viral diarrhoea: a seven year old persistently infected cow a case report. Vet. arhiv 82, 637-643, 2012. ABSTRACT In this report a persistently infected cow was under surveillance for three years. In that period the level of antibodies and level of viremia were determined. Persistent infection was confi rmed in blood serum samples taken at 3 week intervals and tested on the virus antigen by antigen enzyme-linked immunosorbent assay and by inoculation in cell culture, which after incubation was stained by an imunoperoxidase technique. In order to determine the changes in antibody level and virus titer levels, blood samples were collected and tested each six months. Also, data on milk production were collected. The level of viremia was 104.5 throughout the period of the study, and antibodies were not detected by VNT. Milk production was above-average at all times. Average daily milk production over 305 days of lactation in 2009 and 2010 was 25 and 21 litres per day respectively. In the fi rst fi ve months of 2011 daily milk production was 15 litres per day. The cow was taken out of herd at the age of seven years.

Bovine viral diarrhoea virus (BVDV) causes a disease that has a wide range of clinical symptoms in domestic and wild ruminants. It is a major problem in cattle and causes significant economic losses in the cattle industry. The virus infects bovines of all ages and causes both immunosuppression and reproductive, respiratory and digestive disorders. Cattle infected persistently, as a continuing source of the virus and the main factor in transmission of the disease between and among herds, are the main source of BVDV and a primary factor in the epidemiology of the disease. To determine whether a BVDV infection is persistent, two samples should be taken at 3-4 week intervals and tested for the virus antigen. Animal sera, whole blood, organ and ear notch tissue samples can be used for BVDV diagnosis. In ear notch tissue, viral antigen can be detected by an antigen enzyme-linked immunosorbent assay (antigen ELISA), immunohistochemistry (IHC) and reverse-transcription polymerase chain reaction (RT-PCR). This paper describes the development and implementation of an indirect immunofluorescence (IF) method using ear notch tissue samples for diagnosis of cattle infected persistently. Results obtained by this method show that IF is a good alternative to RT-PCR and antigen ELISA and can be a quick and accurate method in diagnosis of BVDV in cattle infected persistently with this virus. (C) 2011 Elsevier B.V. All rights reserved.

In the present study, we report the detection, isolation, molecular characterisation and phylogenetic analysis of bovine herpesvirus type 1 (BoH V-1) in nasal swabs, sera and tissue samples derived from naturally infected cattle. In the spring of 2008, clinical symptoms of the respiratory disease were observed in three Croatian dairy herds. Nasal swabs and sera were collected on the 5(th) and 27(th) days after appearance of symptoms. A tissue sample (lungs) from an eight week old calf was also collected. Specific antibodies against BoHV-1 were not found in sera samples collected after the first sampling, but all sera samples were positive after the second sampling. The cytopathic effect on the bovine embryo kidney (BEK) cells was visible after 72 hours for the nasal swab samples, and after 48 hours for the lung tissue sample. Sensitive PCR assays specific for BHV-1 glycoproteins gB and gC were also performed. All nasal swab samples and the lung sample were positive with both sets of PCR primers. The gC PCR product obtained from one sample from each farm as well as from the lung sample DNA were sequenced. Phylogenetic analysis of gC gene sequence clustered our isolates with BoHV subtype 1.1.

Silvaticna, odnosno sumska bjesnoca ciji su rezervoari uglavnom divlje životinjske vrste na teritoriju Hrvatske prisutna je kontinuirano posljednja tri desetljeca (1977. – 2007.). Tijekom spomenutog perioda u kojem je na tlu RH-a provođena pretraga svih vrsta životinja na bjesnocu, analizom polucenih rezultata utvrdili smo da je lisica (Vulpes vulpes L.) apsolutno najvažniji izvor silvaticne bjesnoce. U svrhu istraživanja pojave i ucestalosti ove opasne bolesti tijekom perioda od trideset godina na podrucju Hrvatske pregledano je 57 776 lisica, od cega je u 12 855 jedinki potvrđena bjesnoca (22, 2%). Posto je epizootiologija silvaticne bjesnoce usko povezana s bioloskim osobitostima i ponasanjem lisica koje su osnovni nositelji sumskog oblika oboljenja, u ovom radu opisali smo i oformili epidemioloski model sirenja silvaticne bjesnoce, uzimajuci u obzir jesensko raseljavanje lisica, radijus kretanja lisica na ''udomacenom'' teritoriju, relativnu gustocu populacije, godisnji priplod i prirast, stopu mortaliteta, godisnji lovni ucinak, koeficijent ucestalosti ugriza i dominantni smjer sirenja bjesnoce s obzirom na GIS kartu. Utvrdili smo da se silvaticna bjesnoca tijekom prve dvije godine pojavljivanja sirila brzinom od 25 km u tri mjeseca u smjeru istok-zapad te brzinom od 18 km/ 3 mj. u smjeru sjever-jug. Najveci broj novoutvrđenih slucajeva bjesnoce po jedinici povrsine zabilježen je u ruralnim podrucjima, uglavnom u okolici manjih naselja, a tijekom vremena bjesnoca se pocela približavati urbanim sredinama. Tijekom prvih deset godina od prodora u Hrvatsku silvaticna bjesnoca se prosirila na gotovo citavu zemlju, a u iducih dvadeset godina trajno se zadržava na nasim prostorima. Najnoviji podaci ukazuju na postojanje enzootije u centralnim dijelovima RH-a, a trend porasta broja na bjesnocu pozitivnih lisica u urbanim podrucjima ukazuje na potrebu provođenja sveobuhvatne i kontinuirane kampanje oralne vakcinacije.

Vec u prva dva mjeseca 2010. godine dijagnosticirano je vise slucajeva bjesnoce u ovaca negoli u svih pojedinih godina tijekom epizootioloskog razdoblja silvaticne bjesnoce u Hrvatskoj.

BACKGROUND:The Orf virus (ORFV) is the prototype of the parapoxvirus genus and it primarily causes contagious ecthyma in goats, sheep, and other ruminants worldwide. In this paper, we described the sequence and phylogenetic analysis of the B2L gene of ORFV from two natural outbreaks: i) in autochthonous Croatian Cres-breed sheep and ii) on small family goat farm.RESULTS:Sequence and phylogenetic analyses of the ORFV B2L gene showed that the Cro-Cres-12446/09 and Cro-Goat-11727/10 were not clustered together. Cro-Cres-12446/09 shared the highest similarity with ORFV NZ2 from New Zealand, and Ena from Japan; Cro-Goat-11727/10 was closest to the HuB from China and Taiping and Hoping from Taiwan.CONCLUSION:Distinct ORFV strains are circulating in Croatia. Although ORFV infections are found ubiquitously wherever sheep and goats are farmed in Croatia, this is the first information on genetic relatedness of any Croatian ORFV with other isolates around the world.

Sismisi su, odmah iza glodavaca, druga najveca skupina sisavaca na planeti – poznato ih je vise od 1000 razlicitih vrsta. Jedini su leteci sisavci, sto im je omogucilo da nasele prakticki sve dijelove svijeta i prilagode se najrazlicitijim ekoloskim uvjetima. Danas u Hrvatskoj obitava 35 vrsta sismisa koji pripadaju trima porodicama. Njihova eurivalentnost na mikroorganizme cini ih pogodnim domacinima za raznovrsne viruse (njih cak 66), ali i za razne druge uzrocnike zaraznih bolesti: bakterije, parazite i gljivice. Kao i svi drugi sisavci, sismisi mogu oboljeti od bjesnoce, te ujedno predstavljaju postojane bioloske rezervoare virusa te fatalne bolesti. Od 1985. u Europi zapocinju sustavna i opsežna istraživanja bjesnoce kod sismisa. Virus bjesnoce dokazan je u velikom broju europskih zemalja, ali je broj sismisa kod kojih je nađen u odnosu na broj pretraženih izuzetno mali, te ne prelazi 2%. Rezultati pretraga sismisa u europskim državama te nepoznavanje situacije u Hrvatskoj potaknuli su Hrvatski veterinarski institut u Zagrebu i strucnjake iz Hrvatskog prirodoslovnog muzeja da zapocnu analizirati dostupne uzorke sismisa na bjesnocu. Do sada su sve obavljene pretrage dale negativne rezultate.