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.
Background 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 ...Pestivirus, within the family Flaviviridae. 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. Findings 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. Conclusions 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.
► 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.
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.
► 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.
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 confirmed 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 10.sup.4.5 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 first five months of 2011 daily milk production was 15 litres per day. The cow was taken out of herd at the age of seven years. Key words: bovine viral diarrhoea, persistent infection, cow, milk production U ovom radu pracene su promjene u razini protutijela i promjene u titra virusa tijekom tri godine u perzistentno zarazene krave. Perzistentna zaraza je dokazana imunoenzimnim testom i izdvajanjem virusa u uzorcima krvnog seruma uzorkovanima dvaput u razmaku od 3 tjedna. Radi dokazivanja promjene u razini protutijela i titra virusa krv je od spomenute krave uzorkovana svakih sest mjeseci. Takoder su prikupljeni i praceni podaci o mlijecnosti krave. Titar virusa bio je 10.sup.4,5 tijekom cijelog istrazivanja pri cemu nije zabiljezena pojava specificnih protutijela. Mlijecnost je bila iznad prosjeka tijekom cijeloga istrazivanja. Prosjecna dnevna proizvodnja mlijeka tijekom 305 dana laktacije 2009. i 2010. godine iznosila je 21 odnosno 25 L, a tijekom prvih 5 mjeseci 2011. godine 15 L. Krava je izlucena iz stada u dobi od sedam godina. Kljucne rijeci: virusni proljev goveda, perzistentna infekcija, krava, mlijecnost
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. Key words: BVDV, immunoperoxidase test, Npro, phylogenetic analysis Od 2007. do 2011. godine na virus virusnog proljeva goveda (VPG) pretrazeni su uzorci pet slezena i 1937 uzoraka seruma podrijetlom iz 9 stada mlijecnih goveda. Imunoperoksidaznim testom (IP) u 13 je uzoraka dokazan necitopatogeni biotip virusa VPG te je potvrdena perzistentna zaraza. Iz svih pet uzoraka slezena izdvojen je citopatogeni virus VPG. U svrhu genotipizacije vlastitih izolata virusa VPG, iz nadtaloga inficiranih stanicnih kultura izdvojena je virusna RNK i umnozena lancanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Umnozeni odsjecci 5'-UTR i gena Npro su potom sekvencirani. Filogenetska analiza hrvatskih izolata virusa VPG pokazala je da svi pretrazeni 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 pretrazenih izolata. Na Npro dijelu genoma utvrdeno je pet aminokiselinskih promjena osebujnih za hrvatske izolate iz podtipa 1f. Kljucne rijeci: virusni proljev goveda, imunoperoksidazni test, gen Npro, filogenetska analiza
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.
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