To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular ...methods for diagnostics of brucellosis in domestic animals.
We serologically tested 42,785 cattle serums, 22,686 sheep and goat serums, and 28520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10,173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples.
B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 flocks in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties.
In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.
The antimicrobial susceptibility of coagulase-negative staphylococci isolated from spontaneously fermented sausages was assessed using both traditional and molecular methods. Isolates were tested for ...sensitivity to vancomycin, ampicillin, erythromycin, tetracycline, gentamycin and oxacillin by the disk diffusion method and quantitative-qualitative epsilometer test. PCR was used for the detection of resistance genes mecA, ermB, tetK and tetM. The identified coagulase-negative staphylococci were Staphylococcus epidermidis (69 %), S. capitis (5 %) and S. warneri (2.5 %). S. epidermidis showed a high rate of phenotypical resistance to tetracycline and erythromycin (44.4 % of strains). Molecular evaluation of resistance determinants revealed tetK or tetM genes in eight S. epidermidis strains. Although S. epidermidis is not classical food poisoning bacteria, its presence in food could be of public health significance due to the possible spread of antimicrobial resistance determinants. Our findings implicate that spontaneous meat fermentation could result in products with a potential hazard to consumers.
In a recent lambing season (2012/2013), the seroprevalence of ovine chlamydiosis was monitored in small ruminant abortion cases in Croatia. Blood samples of 93 sheep and 69 goats were examined. In ...addition, 50 sheep and 61 goat samples were tested using molecular methods. Furthermore, 14 sheep blood samples, one goat blood sample and one sheep placenta sample from Bosnia and Herzegovina (BIH) were also tested as a part of inter-laboratory cooperation. Overall high seroprevalence was detected in sheep, 19.6% with the ELISA IDEXX kit and 20.5% with the ClVTEST kit. Seroprevalence in goats was 11.4%. In BIH, four sheep and one goat blood sample were seropositive for chlamydiosis. The disease causing agent, Chlamydia abortus (C. abortus) was confirmed using molecular methods in two sheep flocks in continental Croatia and in one sheep flock in BIH. In this study, C. abortus infection in sheep was identified for the first time in Croatia using species specific molecular methods. Ovine chlamydiosis is present in national sheep and goat flocks in Croatia and BIH. Thus should be subject to ongoing controls in the case of abortion. A combination of serological and molecular methods should be used for optimal laboratory diagnostics of C. abortus.
An epizootiological field study on tick-borne protozoan infections in foxes (
Vulpes vulpes) was carried out in different parts of Croatia. Spleen samples of 191 carcasses of red foxes killed in ...sanitary hunting, were examined for the presence of hematozoa by polymerase chain reaction (PCR) and subsequent sequencing. The investigation revealed four species of hematozoa in 57 foxes (30%), namely
Theileria annae,
Theileria sp. 3182/05 and
Hepatozoon canis.
T. annae was found in 10 foxes (5%),
Theileria sp. 3182/05 in a single animal (1%),
H. canis in 44 (23%) and
Hepatozoon sp. was detected in two foxes (1%).
T. annae and
H. canis were distributed through all the studied regions, while
Theileria sp. 3182/05 and
Hepatozoon sp. were restricted to the Zagreb and Zagorje, and Istria regions, respectively. Detection of
T. annae in all regions of Croatia indicates the presence of the natural cycle of the parasite and raises the possibility of other vectors other than the proposed
Ixodes hexagonus.
This study’s objective was to estimate the seasonal occurrence of aflatoxin M1 (AFM1) in cow’s milk between winter 2016 and winter 2022 and to assess dietary exposure and risk assessment for the ...adult Croatian population. In total, 5817 cow milk samples were screened for AFM1 concentrations using the enzyme immunoassay assay (ELISA). For confirmation purposes of AFM1 concentration above the European Union maximum permitted level (MRL), ultra high-performance liquid chromatography with tandem mass spectrometry was performed. In 94.7% of milk samples, AFM1 levels were below the detection limit (LOD) of the ELISA test. For 3.47% of samples, the AFM1 was between the LOD and MRL values. Only 1.87% of all samples exceeded the MRL. The mean value of elevated AFM1 in different seasons ranged between 59.2 ng/kg (autumn 2017) and 387.8 ng/kg (autumn 2021). The highest incidences of positive AFM1 were determined in autumn and winter and the maximum (6.4%) was in winter 2019/2020. The largest percentage of positive samples (69.7%) was found in central Croatia. The estimated daily intakes for positive samples ranged between 0.17 and 2.82 ng/kg body weight/day. Risk assessment indicated a high level of concern during autumn and winter, especially for consumers of large amounts of milk.
Aflatoxin M1 (AFM1) concentrations were measured in bulk cow milk samples from eastern Croatia, and in cow, goat, sheep and donkey bulk milk samples from other parts of Croatia during the period ...July–September 2013. AFM1 levels in milk were measured in the ranges (ng/L): cow 3.65–162.3 (eastern Croatia) and 2.69–44.9 (other regions of Croatia); goat 2.78–40.8; sheep 2.11–5.87; donkey 3.43–10.4. The concentration of AM1 exceeded the EU MRL in 6.7% of cow milk samples from eastern Croatia. The highest level measured was 162.3 ng/L. AFM1 levels exceeded the LOQ value (23.2 ng/L) in only 59 samples of cow milk and two samples of goat milk of the total 402 samples analysed. A significant difference was found between the mean AFM1 concentrations of cow milk from eastern and other regions of Croatia (P < 0.05). The elevated AFM1 levels in cow milk from eastern Croatia indicate the use of contaminated supplementary feedstuff in some farms during the study period.
•The AFM1 levels in cow, goat, sheep and donkey milk from Croatia were determined.•In 15.2% of samples AFM1 concentrations exceeded quantification limit of the method.•In 6.7% of cow milk from eastern Croatia AFM1 levels exceeded the EU limit.•A significant difference between the cow milk from eastern and other part of Croatia were found.
In order to detect thermotolerant
spp., 241 samples of fresh chicken meat, at retail in Croatia, were analysed according to a standard method, followed by biochemical test and molecular polymerase ...chain reaction/restriction enzyme analysis for exact species determination.
spp. prevalence was 73.86%.
and
were isolated from 53.53 and 15.35% of the samples, respectively. In 4.98% of isolates thermotolerant
spp. were not determined. The multilocus sequence typing method was used to evaluate genetic diversity of eight
and four
isolates. To our knowledge, these results of genotyping provided the first data on the presence of sequence types (STs) and clonal complexes (CCs) of
and
.
isolates in Croatia. By applying the multilocus sequence typing, a new allele of
gene locus was discovered and marked
508. The
.
ST 6182 and
.
ST 6183 genotypes were described for the first time, and all other identified genotypes were clustered in the previously described sequence types and clonal complexes. These findings provide useful information on the prevalence and epidemiology of
and
in Croatia.
► 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.
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