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Bovine brucellosis is diagnosed by official tests, such as Rose Bengal plate test (RBPT) and Complement Fixation test (CFT). Both tests detect antibodies directed against the lipolysaccharide (LPS) of Brucella cell wall. Despite their good sensitivity, those tests do not discriminate between true positive and false positive serological reactions (FPSR), the latter being generated by animals infected with other Gram negative microorganisms that share components of Brucella LPS. In this study, an antigenic extract from whole Brucella melitensis B115 strain was used to set up an ELISA assay for the serological diagnosis of bovine brucellosis. A total of 148 serum samples from five different groups of animals were tested: Group A: 28 samples from two calves experimentally infected with Yersinia enterocolitica O:9; Group B: 30 samples from bovines infected with Brucella abortus; Group C: 50 samples from brucellosis-free herds; Group D: 20 samples RBPT positive and CFT negative; Group E: 20 samples both RBPT and CFT positive. Group D and Group E serum samples were from brucellosis-free herds. Positive reactions were detected only by RBPT and CFT in calves immunized with Y. enterocolitica O:9. Sera from Group B animals tested positive also in the ELISA assay, whereas sera from the remaining groups were all negative. The results obtained encourage the use of the ELISA assay to implement the serological diagnosis of brucellosis.

The Ohrid trout (Salmo letnica) is an endemic species of trout found only in Lake Ohrid, in the Balkan republics of North Macedonia and Albania, where it is a species of significant economic and nutritional interest. Considering the importance of chemical risk assessment in fish for human consumption set out by European and international legislation and the risk of extinction of the Ohrid trout, the aim of the present study was to evaluate the safety of Ohrid trout flesh for human consumption by determining the heavy metal concentration in different organs through atomic absorption spectrometry (AAS). Heavy metal concentrations differed significantly in different organs (P

The results of a study designed to evaluate the performance of an in-clinic test for the detection of canine parvovirus (CPV) are reported. A total of 150 faecal samples collected from dogs with acute diarrhoea were tested using the in-clinic test, a haemagglutination assay (HA) and a real-time PCR assay for CPV detection, quantification and characterisation. CPV was detected in 66, 73, and 101 faecal samples by in-clinic, HA and PCR testing, respectively. The relative sensitivity and specificity of the in-clinic test were 86.3% and 96.1%, respectively, when the test was compared to HA, and 65.3% and 100%, respectively, when compared to real-time PCR. The sample distribution according to the virus type was CPV-2a, n = 44; CPV-2b, n = 11; CPV-2c, n = 44, CPV-2, n = 2, as determined by minor groove binder probe assays and/or sequence analysis. The percentage of positive in-clinic tests was 70.5% for CPV-2a, 72.7% for CPV-2b and 75.0% for CPV-2c ( P > 0.05). Using real-time PCR as the reference standard for CPV detection, the in-clinic test was more specific than HA and had comparable sensitivity to HA, demonstrating detection rates similar to those previously observed for other rapid in-clinic tests. The in-clinic test was also able to detect all CPV types at equivalent rates.

Given that changes in consumer food behaviours have led to an increase in the demand for pre-cut ready-to-eat (RTE) vegetables, and that few data are currently available on the occurrence of Arcobacter spp. in such foods, the aim of the present study was to assess the occurrence of Arcobacter spp. that carry virulence-associated genes on pre-cut RTE vegetables, using cultural and molecular methods. Arcobacter was detected using biomolecular identification methods in 44/160 (27.5%) of the samples, of which 40/44 (90.9%) isolates corresponded to A. butzleri and 4/44 (9.1%) to A. cryaerophilus. Studying the incidence of 9 virulence-associated genes revealed the widespread distribution of these genes among the Arcobacter isolates tested. The results obtained in our research provided plenty of information on the health risks associated with the direct consumption of raw vegetables, and highlight the need to implement further studies at each level of the production chain, in order to obtain further information to help protect human health.

Aims: This study evaluated the application of polymerase chain reaction–enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. Methods and Results: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. Conclusions: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. Significance and Impact of the Study: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.

Recently, canine coronavirus (CCoV) strains with putative recombinant origin with porcine transmissible gastroenteritis virus (TGEV) were shown to be widespread in Europe. In this study, a killed vaccine against TGEV-like CCoV strains, included in the new subtype CCoV-IIb, was developed through inactivation with betapropiolactone and emulsification with MF59™ adjuvant. Safety, immunogenicity and efficacy of the developed vaccine were evaluated in vivo. Five 10-week-old beagle pups were administered (three weeks apart) two vaccine doses, whereas two animals served as unvaccinated controls. The vaccine was shown to be safe as no local neither systemic reactions were observed after first and second dose administration. Serum antibodies against CCoV were detected in vaccinates starting from study day 14 (by enzyme-linked immunosorbent assay) or 28 (by virus neutralisation test). Subsequent challenge with virulent CCoV-IIb resulted in the development of mild gastroenteric disease in control pups, whereas vaccinates did not display clinical signs. Faecal shedding of the challenge virus occurred in both treatment groups, but vaccinated dogs were found to shed very low viral titres in comparison to controls. The developed vaccine may help control the CCoV-IIb-induced disease (and active virus circulation) in environments, such as kennels and shelters, where the pathogenic potential of this virus is greater as a consequence of predisposing factors and concurrent infections.

Summary This study provides data on the prevalence of potentially pathogenic Bacillus cereus in foods from catering kitchens by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. B. cereus was detected in 72/250 (28.8%) food samples. Specifically, B. cereus was highlighted in 34/74 (45.9%) pastries, 16/40 (40%) rice samples, 4/38 (10.5%) potato meals, 6/54 (11.1%) mozzarella samples and 12/44 (27.3%) meat meals. PCRs aimed at the hbl (C, D, A, B), nhe (A, B, C), bceT and cytK genes demonstrated a widespread distribution of the toxin-encoding genes among B. cereus isolates. The results highlight the frequent failure of control measures in catering kitchens and the need for intensive and continuous monitoring in order to assess the human health risk, as proposed by Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs.

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species.

The correct diagnosis of canine minute virus is critical in dog breeding. In this study, the Bland Altman test was used to compare the performance of two susceptibility-testing methods, namely polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). The agreement between IFA and PCR in monocytes revealed a mean difference of −1752.16 with 95% confidence and an interval ranging from −3229.80 to −274.53 (SD = 2325.62). The agreement between IFA and PCR in Walter Reed canine cells (WRCC) revealed a mean difference of −2396.55 with 95% confidence and an interval ranging from −3774.63 to −1018.48 (SD = 2168.93). The Bland Altman test confirmed the overall accuracy of PCR vs IFA and the plot showed that all points were not randomly arranged in the range of average ±1.96 × SD of the differences.

Dermatitis caused by Malassezia spp., one of most common skin disease in dogs, requires prolonged therapy and/or high doses of antifungal agents. In the present study, the antifungal susceptibility of M. pachydermatis to ketoconazole (KTZ), fluconazole (FLZ), itraconazole (ITZ), posaconazole (POS) and voriconazole (VOR) was evaluated in vitro using both CLSI reference broth microdilution (CLSI BMD) and E-test. A total of 62 M. pachydermatis strains from dogs with and without skin lesions were tested. M. pachydermatis strains were susceptible to ITZ, KTZ and POS using both test methods, with the highest MIC found in tests of FLZ. Essential agreement between the two methods ranged from 87.1% (VOR) to 91.9% (ITZ), and categorical agreement from 74.2% (FLZ) to 96.8% (ITZ). Minor error discrepancies were observed between the two methods, with major discrepancies observed for KTZ. A higher MIC(50) value for FLZ was noted with M. pachydermatis genotype B. The MICs(50) of M. pachydermatis genotype B for KTZ, VOR and POS were higher in isolates from dogs with skin lesions than those in isolates from animals without skin lesions. The results suggest a link between genotypes of M. pachydermatis and in vitro drug susceptibility. The categorical agreement for both E-test and CLSI BMD methods found in this investigation confirms the E-test as a reliable diagnostic method for routine use in clinical mycology laboratories.

The microdilution antifungal method (CLSI BMD, M27-A3) was used for testing the antifungal susceptibility of Malassezia species. However, optimal broth media that allow sufficient growth of M. pachydermatitis and produce reliable and reproducible MICs using the CLSI BMD protocol are yet to be established. In this study, the susceptibility of M. pachydermatis isolates to ketoconazole (KTZ), itraconazole (ITZ) and fluconazole (FLZ) was evaluated in vitro by the CLSI BMD test using Christensen's urea broth (CUB) and mRPMI 1640 containing lipid supplementation, Sabouraud dextrose broth with 1% tween 80 (SDB), and Dixon broth (DXB). A FLZ-resistant M. pachydermatis was generated in vitro and tested under the same conditions. A good growth of M. pachydermatis incubated for 48 and 72 h, respectively, was observed in CUB, SDB and DXB and not in mRPMI 1640 (p0.001). No statistically significant differences were detected between the MIC values registered after 48 h and 72 h incubation. ITZ displayed lower MIC values than KTZ and FLZ regardless of the media employed. A large number of FLZ-resistant Malassezia strains (86.6%) was observed using DXB. A MIC64 mg/L was observed only when the FLZ-resistant M. pachydermatis isolate was tested in SDB. Based on the results obtained herein, culture in SDB, stock inoculum suspensions of 1-5 × 10(6)CFU/ml, and an incubation time of 48 h are proposed as optimal conditions for the evaluation of the in vitro antifungal susceptibility of M. pachydermatis using a modified CLSI BMD protocol.

Background In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field investigation. Results The results indicated that, in contrast to the classic method, the GABRI method was able to detect B.anthracis in all contaminated samples. The GABRI method produces a more sensitive measure of anthrax spore presence significantly different from the standard method. In particular, the latter is more sensitive to the presence of normal soil contaminants. Conclusion The main feature of the GABRI method is its ability to strongly reduce the presence of the environmental contaminants, which being much more numerous than B. anthracis tend to inhibit their germination and growth making it extremely difficult to visualize any colonies. The reduction of the microbial environment also allows one to be able to culture and test a larger quantity of potentially contaminated soil and to isolate B. anthracis when the spores are present in very low concentrations in the soil.