► 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.
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.
•We have genotyped Coxiella burnetii strains for the first time in Croatia.•For the genotyping we used MLVA.•We have identified 13 novel C. burnetii genotypes that appear to be unique to Croatia.
...Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping.
The aim of the study was to investigate annual and regional differences in the level of aflatoxin B1 (AFB1) in grains and dairy cattle feed. Maize (n = 972), wheat (n = 201), barley (n = 147), oat ...(n = 136), grain mixtures (n = 168), and dairy cattle feed (n = 325) were sampled from 2009 to 2013 on different farms and in different farm factories situated in four Croatian regions. The samples were analysed for AFB1 using the validated ELISA immunoassay. AFB1 was determined in 16.4% of all investigated samples, among which maize was proven to be the most contaminated, with 21.7% of the samples recovered during 2013 harbouring AFB1 in concentrations over the permissible ones. Levels higher than permitted were observed in 17.9% and 12.3% of grain mixtures and dairy cattle feed, respectively, whereas concentrations of AFB1 determined in other crops throughout the investigated period met the stipulated requirements. The results revealed the AFB1 occurrence to be significantly (p < 0.05) dependent on the cultivation region, with the highest levels generally found in maize harvested in 2013 and consequently in grain mixtures and cattle feed that can most likely be associated with climatic conditions as the most critical factor for mould formation, and thus also AFB1 production.
•Aflatoxin B1 in grains and feed from dairy farms over a 5-year period was determined.•ELISA method was implemented.•Contamination levels were dependent on the cultivation region and the year of sampling.•The highest levels were generally found in maize.•Elevated levels were consequently determined in grain mixtures and feed.
•MLVA was used to genotype 127 B. suis strains from SE Europe.•Forty-one novel genotypes were deposited into the Brucella2012 database.•B. suis SE Europe strains tested are regionally specific and ...some are unique.•Typing issues with Brucella19 locus were affirmed.•Swine brucellosis in SE Europe should be researched further and in more detail.
Porcine brucellosis is a common bacterial zoonosis which can cause significant financial losses. Its diverse and often complicated factors have hampered efforts to control disease spread.
The aim of the study was to assess the epidemiological situation of porcine brucellosis primarily in Croatia and its relationship to genotypes present in other, mostly European countries. One hundred and seven Brucella suis strains isolated from swine, hares, cattle, humans, wild hares, a wild boar and a mare originating mainly from Croatia (112), but also a few from Slovenia, Bosnia and Herzegovina, Serbia and Macedonia (15) were tested using classical microbiological testing, Bruce-ladder, RFLP, Multiplex-suis and genotyped using multi-locus variable-number tandem repeat analysis (MLVA).
We determined 43 Brucella suis genotypes. Strains were grouped according to phylogenetic and geographic relationships, revealing both regional specificity and uniqueness and suggesting possible sources and modes of spread among animals. Our study also confirmed problems with Bruce19 locus that may hinder comparisons of new types with those in the international database.
Forty-one novel genotypes were identified and deposited into the international database. Our study supports the idea of wild animals as a source of disease in domestic animals and also gives evidence to hypothesis of cross-border animal trafficking between former Yugoslavian countries. It also highlights the need to expand such research across more of southeast Europe, especially to countries with poorer social and economical situation in order to prevent a realistic outbreak and for better understanding of the biology of this pathogen.
A total of 3543 raw cow milk samples were collected in three regions of Croatia: western, eastern and other regions during four seasons. Samples were measured for aflatoxin M1 (AFM1) concentrations ...using the enzyme immunoassay method. Elevated levels (>50 ng/kg) of AFM1 were analysed by validated liquid chromatography with triple quadruple mass spectrometry (LC-MS/MS). The limits of detection (LOD) and quantification (LOQ) of the LC-MS/MS method were 7.3 and 28 ng/kg, respectively. The mean AFM1 levels measured in the three regions over four seasons were in the ranges (ng/kg): eastern Croatia 7.25–26.6; western Croatia 5.91–9.26; other regions of Croatia 7.17–13.6. The highest incidence of samples exceeding the EU MRL (50 ng/kg) of 9.32% was measured in autumn (October–December) in the eastern region. Only eight samples were found to exceed the EU MRL in winter. The highest AFM1 levels were measured in December (764.4 ng/kg) and January (383.3 ng/kg). Elevated AFM1 levels were found in summer in only four samples from the western and other regions, and two samples in the eastern region. This can be attributed to localized and random usage of contaminated feed for dairy cows in those regions. The much lower incidence of elevated AFM1 in comparison to a previous study showed that the outbreak of the crisis due to elevated AFM1 levels in 2013 resulted in a more careful approach to the control of supplementary feedstuff for lactating cows.
•AFM1 concentrations in milk were monitored during one year period in Croatia.•During autumn 9.32% samples exceeding the EU MRL in the eastern region.•In other seasons there were no differences in AFM1 levels between three regions.•Only a few samples with elevated concentrations were found during the summer.
This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of ...treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 μg/kg; detection capability, 0.56 μg/kg; limit of detection, 0.04 μg/kg; limit of quantification, 0.15 μg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 μg/kg in egg yolks and 166.5 μg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 μg/kg) and on day 37 in egg yolks (0.06 μg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.
Aflatoxin M1 (AFM1) concentrations were determined in raw and UHT cow milk samples collected in different regions of Bosnia and Herzegovina and Croatia during the autumn months of 2014. The mean AFM1 ...levels in the raw milk samples were (ng/kg): 6.22 in Bosnia and Herzegovina, 5.65 in Croatia. In all except one milk sample, AMF1 levels were below the LOQ value of 34.2 ng/kg (ELISA method). In four milk samples, AFM1 concentrations exceeded the EU MRL of 50 ng/kg. Samples were subjected to LC-MS/MS analysis which confirmed elevated values determined by ELISA. Elevated levels were in the range 56.6–132.6 ng/kg. Two positive milk samples from Bosnia and Herzegovina originated from Una Sana Canton, two from Croatia from eastern Croatia. The highest AFM1 levels of 132.6 ng/kg was measured in milk from eastern Croatia. In 214 samples of processed UHT milk from Bosnia and Herzegovina and Croatia, AFM1 ranged from 2.29 ng/kg to 21.4 ng/kg, all below the LOQ value. AFM1 exceeded the EU MRL value in only 0.62% of milk samples, indicating the sporadic use of contaminated feedstuff at farms in both countries.
•Raw and UHT milk were collected in Bosnia and Herzegovina and Croatia.•During autumn 2014 only 0.62% raw milk exceeded the maximum EU limit for AFM1.•Two samples from Bosnia and Herzegovina and two from Croatia exceeded EU limit.•Four elevated AFM1 concentrations were confirmed by LC-MS/MS.•In all UHT milk samples AFM1 levels were below LOQ of the ELISA method.
Bakterije
mikroorganizmi koji za svoj rast u okolišu, kao i u laboratorijskim uvjetima trebaju mikroaerofilne uvjete. Za rutinsku analitiku klasičnim metodama primjerena je primjena priznatih ...standardiziranih protokola i propisanih visoko selektivnih tekućih i/ili krutih hranjivih podloga. Nakon porasta karakterističnih kolonija radi određivanja vrste kampilobaktera primjenjuju se mikroskopski, fenotipski, biokemijski, imunozimski testovi, rezistotipizacija i/ili metoda masene spektrometrije. Za razlikovanje sojeva kampilobaktera postoje dvije klasične metode serotipizacije. Do sada je razvijen znatan broj PCR protokola usmjerenih prema otkrivanju Campylobacter roda, vrste ili podvrste, bilo iz uzoraka hrane, okoliša ili izmeta. Glavne prednosti PCR metoda su u njihovoj brzini, osjetljivosti i specifičnosti, a nedostatak PCR metodologije je u tome što ne razlikuje žive od mrtvih stanica, zbog čega se razvijaju novi protokoli bazirani na RT-PCR, NASBA i QRT- PCR molekularnim metodama.
