Molecular methods are increasingly being utilized for accurate identification of ticks (Acari: Ixodidae), especially in cases of morphologically highly similar species. In this study, we performed ...molecular research of the tick fauna in Croatia using DNA barcoding method. Ticks were sampled in three biogeographical regions and thirteen species were recorded: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma marginatum, Ixodes frontalis, Ixodes hexagonus, Ixodes kaiseri, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus sanguineus s.l. and Rhipicephalus turanicus. Ixodes kaiseri is for the first time recorded in the fauna of Croatia. Of the thirteen hard tick species analyzed in this study, pathogens from different groups (bacteria, protozoa and viruses) have been detected in eight species in Croatia so far. For the important vector species R. sanguineus s.s., new distributional data for Croatia are given. The standard COI barcoding region was amplified, and the sequences were analyzed by species delimitation methods together with the sequences of conspecific and congeneric species from the public BOLD database. Our specimens of H. punctata represent a new, genetically distinct MOTU. A brief overview of the available public DNA barcoding data for Ixodidae is presented, highlighting the need for an integrative approach for the clarification of the taxonomic status of problematic Ixodid taxa. The results provide a basis for the establishment of a molecular data platform for the Ixodidae of the Croatian fauna.
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.
In a total of 1536 blood serum samples analysed by ELISA, antibodies for IAV nucleoprotein (NP) were detected in 30.3%. Results from HI show that the most common subtype of swIAV in the Croatian pig ...population was H1N1 (44.6%), followed by H3N2 (42.7%) and H1N2 (26.3%). Antibodies to at least one subtype were detected in 62.19% of blood serum samples. Detection of swIAV antigen was performed by IHC and detected in 8 of 28 lung samples collected post-mortem. The matrix (M) gene was detected in nine of one hundred and forty-two lung tissue samples and in seven of twenty-nine nasopharyngeal swabs. Phylogenetic analysis of amplified HA and NA gene fragments in Croatian isolates suggests the presence of swIAV H1avN1av.
West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our ...study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions.
BARBIC, LJ., V. SAVIC,K. KOVACEVIC, J. KAPETAN, V. STEVANOVIC, S. KOVAC, S. HADINA, Z. MILAS, V. STARESINA, N. TURK, M. PERHARIC, J. MADIC: Outbreak of equine influenza in Croatia in 2015 and post ...outbreak epidemiological situation. Vet. arhiv 88, 437-451, 2018. In March 2015, a few days after a major horse fair event in Bjelovar, Croatia, an equine influenza outbreak began and in the days that followed spread to more than 20 stud farms in the continental part of Croatia. The epidemiological investigation showed the importance of the national and international movement of asymptomatic carrier animals as a major risk factor for the introduction of the equine influenza virus and its spread in the naive population. Molecular characterization and phylogenetic analyses confirmed that the EI outbreak was caused by an imported viral strain of the H3N8 subtype, phylogenetically similar to recent European strains belonging to Florida sublineage clade 2. The post-outbreak equine influenza seroprevalence in continental Croatia, based on ELISA testing, was 12.3% and varied between 1.1% and 32.6% on a county level. The highest seroprevalence in counties with predominantly sport and leisure horses highlighted animal management as a principal risk factor for equine influenza infection. On the other hand, variations in haemagglutination inhibition titres in the tested serum samples suggested different times of infection acquisition and unreported outbreaks of equine influenza in continental Croatia after the 2015 outbreak. Vaccination coverage, even after the large outbreak in 2015, was still below 10%, which suggests the lack of education of horse owners and represents a high risk for further outbreaks. In conclusion, influenza is a wide spread infection with multiple, often unrecorded, outbreaks in continental Croatia. A high risk of further outbreaks is present due to low vaccination coverage, an increase in the sport and leisure horse population and the intensive movement of those animals. In order to prevent further outbreaks of equine influenza in Croatia it is necessary to introduce adequate surveillance and mandatory vaccination at least for the sport and leisure horse population where there is international transport of animals. Key words: equine influenza; outbreak; international transport; seroprevalence; ELISA; IHA; vaccination coverage; Croatia
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.
► 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.
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.
In the period from 2015 to 2020, an entomological survey for the presence of West Nile virus (WNV) and Usutu virus (USUV) in mosquitoes was performed in northwestern Croatia. A total of 20,363 ...mosquitoes were sampled in the City of Zagreb and Međimurje county, grouped in 899 pools and tested by real-time RT-PCR for WNV and USUV RNA. All pools were negative for WNV while one pool each from 2016 (
), 2017 (
complex), 2018 (
complex), and 2019 (
complex), respectively, was positive for USUV. The 2018 and 2019 positive pools shared 99.31% nucleotide homology within the USUV NS5 gene and both clustered within USUV Europe 2 lineage. The next-generation sequencing of one mosquito pool (
complex) collected in 2018 in Zagreb confirmed the presence of USUV and revealed several dsDNA and ssRNA viruses of insect, bacterial and mammalian origin.
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