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.
► Fluorescent antibody virus neutralisation test was modified. ► Muscle extract and thoracic liquid as samples in the follow-up of fox oral vaccination campaigns against rabies were evaluated. ► ...Cytotoxic effect on cells is completely prevented.
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 1h 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.
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.
Serologic evidence of exposure to porcine parvovirus (PPV) in the wild boar (Sus scrofa) in Croatia was investigated. Serum samples from 219 wild boars captured during 2003 from 12 different ...locations in the Republic of Croatia were tested by using a commercial enzyme-linked immunoassay (ELISA) and a hemagglutination inhibition (HI) test. Antibodies to PPV were detected in 91 (41.6%) of tested samples and positive results were detected in wild boar from all sample locations. Adults had a significantly higher prevalence (70%) than juveniles (31%; P<0.01). Our results indicate that wild boar populations throughout the Republic of Croatia are exposed to PPV.
► Indirect immunofluorescence (IIF) assay on ear notch tissue is applied for BVDV diagnosis of PI cattle. ► Compared to RT-PCR, the IIF had a sensitivity and specificity of 100%. ► The ...immunofluorescence signal was mainly detected within interfollicular space and in some samples it was considerably inferred within hair follicle.
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.
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 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.
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.
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.
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 less than 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.
Enzyme linked immunosorbent assays (ELISAs) and a nested polymerase chain reaction after reverse transcription (RT-PCR) were used for the detection of the Chinese strain (C strain) of classical swine ...fever virus (CSFV) in blood and tissue samples of experimentally inoculated piglets. One group of 10 piglets was inoculated with C strain material from rabbits and a second one with material from infected minipig kidney (MPK) cell culture. Tested blood samples were taken on the day of inoculation as well as on days 2, 4, 6, 8, 10, 13 and 16. Samples of spleen, tonsil and brain tissue were collected from piglets on days 6, 8, 10, 13 and 16 and tested for glycoprotein E
RNS and protein NS2-3 using commercially available ELISA kits. E
RNS and NS2-3 were detected earlier in blood samples of piglets inoculated with the C strain propagated in a cell culture. Regardless of propagation the presence of the viral E
RNS and NS2-3 was detected in spleen and tonsil samples simultaneously. The C strain propagated in a cell culture was found in only one brain sample, whereas, the virus propagated in rabbits was detected in 70% of the brain samples. For the detection of the CSFV RNA in blood samples, a part within the 5′ non-coding region was amplified. The differences in the results gained by antigen detection in blood samples decreased when nested RT-PCR was used.
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