Filogenetska analiza hrvatskih izolata virusa virusnog proljeva goveda.

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-1b and 7 with BVDV- 1f 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-1f isolates.Od 2007. do 2011. godine na virus virusnog proljeva goveda (VPG) pretraženi su uzorci pet slezena i 1937 uzoraka seruma podrijetlom iz 9 stada mliječnih goveda. Imunoperoksidaznim testom (IP) u 13 je uzoraka dokazan necitopatogeni biotip virusa VPG te je potvrđena perzistentna zaraza. Iz svih pet uzoraka slezena izdvojen je citopatogeni virus VPG. U svrhu genotipizacije vlastitih izolata virusa VPG, iz nadtaloga inficiranih staničnih kultura izdvojena je virusna RNK i umnožena lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Umnoženi odsječci 5’-UTR i gena Npro su potom sekvencirani. Filogenetska analiza hrvatskih izolata virusa VPG pokazala je da svi pretraženi izolati pripadaju genotipu 1; 11 izolata svrstano je u podtip 1b, a 7 u 1f. Filogenetska analiza na temelju spojene 5’-UTR/Npro genske sekvencije potvrdila je rezultate genotipizacije pretraženih izolata. Na Npro dijelu genoma utvrđeno je pet aminokiselinskih promjena osebujnih za hrvatske izolate iz podtipa 1f

Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

<p>Abstract</p> <p>Background</p> <p>Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus <it>Pestivirus</it>, within the family <it>Flaviviridae</it>. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described.</p> <p>Findings</p> <p>Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells.</p> <p>Conclusions</p> <p>Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.</p

Leptospiral antibodies in red foxes (Vulpes vulpes) in northwest Croatia

In 2000 sera from 59 red foxes (Vulpes vulpes) from northwest Croatia were tested for antibodies against 12 Leptospira interrogans serovars: grippotyphosa, sejroe, australis, pomona, canicola, icterohaemorrhagiae, tarassovi, saxkoebing, ballum, bataviae, poi and hardjo. Using the microscopic agglutination test in 34 (57.6%) of the 59 sera, leptospira antibodies were found for 11 serovars, mainly for the serovar australis, then (in decreasing order) for the serovars sejroe, icterohaemorrhagiae, grippotyphosa and saxkoebing. The results showed a strong correlation with previous results of seroepidemiology and isolation of leptospira from small mammals in northwest Croatia. The role of the red fox as a maintaining leptospira reservoir in the natural foci of leptospirosis is still a matter of question and should be further investigated. Key words: red fox, Vulpes vulpes, leptospira, natural foci, Croati