Background: The bacterial species S. aureus is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by S. aureus ranges from 2% to more than 50%, and it causes 10–12% of all cases of clinical mastitis. Aim: The objective was to analyze 237 strains of S. aureus isolated from the milk of cows with subclinical mastitis regarding the spa, mecA, mecC and pvl genes and to perform spa and multi-locus sequence typing (MLST). Methods: Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of spa and seven housekeeping genes. Results: The spa fragment was present in 204 (86.1%) of strains, while mecA and mecC gene were detected in 10 strains, and the pvl gene was not detected. Spa typing successfully analyzed 153 tested isolates (64.3%), confirming 53 spa types, four of which were new types. The most frequent spa type was t2678 (14%). MLST typed 198 (83.5%) tested strains and defined 32 different allele profiles, of which three were new. The most frequent allele profile was ST133 (20.7%). Six groups (G) and 15 singletons were defined. Conclusion: Taking the number of confirmed spa types and sequence types (STs) into account, it can be concluded that the strains of S. aureus isolated from the milk of cows with subclinical mastitis form a heterogenous group. To check the possible zoonotic potential of isolates it would be necessary to test the persons and other livestock on the farms.

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

The aim of this study was to determine the effects of dietary vibroactivated clinoptilolite supplementation on the intramammary microbiological findings in dairy cows, causative agents of potential intramammary infection, and their sensitivity to antibiotics. Cows (n = 78) were randomly divided into two groups: CPL-treated group that received clinoptilolite (CPL) in-feed (n = 38), i.e., 50 g natural powdered zeolite CPL, twice daily from the seventh month of pregnancy to 75 days after calving, and the control group (CON) of untreated cows (n = 40). Milk samples were taken from each cow on days 7, 25, 45, and 75 postpartum. The following causative pathogens were isolated in 86 udder quarters: Staphylococcus aureus in 5.81% of positive samples, Staphylococcus spp. 9.32%, coagulase-negative Staphylococcus (CNS) 22.09%, Streptococcus uberis 13.95%, Streptococcus agalactiae 1.16%, Streptococcus sp. 3.49%, Escherichia coli 8.13%, Enterococcus spp. 6.98%, Corynebacterium spp. 11.63%, Pasteurella sp. 10.47%, Serratia spp. 2.33%, and Arcanobacterium pyogenes, Citrobacter sp., Prototheca sp., and yeasts each in 1.16% of samples. Additionally, 3.87% of environmental microflora samples (n = 47) and 89.06% of udder samples (n = 1083) were bacteriologically negative. The most effective antibiotics were cefoperazone and amoxicillin-clavulanic acid, while cloxacillin and tetracycline were the least effective antibiotics in both groups. In the clinoptilolite supplemented (CPL) group (n = 38) of 14 cows, nine causative agents of mastitis were isolated in 27 quarters, while in the control (CON) group (n = 40) of 24 cows, 13 causative agents of mastitis were isolated in 59 quarters. Cows from the CON group had a 1.96 times higher risk of intramammary infection than cows from the CPL group during the observation period (odds ratio = 1.96, p = 0.0031; 95% CI = 1.2570–3.0770).

Studies of polyphenols in various natural products have shown their variability and often low amounts. Thus, we aimed to investigate the bioaccessibility of the main polyphenolic groups extracted from two poplar types of propolis collected in continental Croatia. The propolis complex, in the form of water-soluble powder of propolis extract with the carrier 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), was obtained by applying ultrasonic-assisted extraction followed by lyophilization. The in vitro gastrointestinal digestion was conducted on the propolis complex extracts and a mixture of standards (pinocembrin, galangin, chrysin, caffeic acid, and caffeic acid phenylethyl ester). Fractions from the oral, gastric, and intestinal phase samples were analyzed spectrophotometrically by standard methods used to determine the content of total phenols (TPs), flavonols/flavones (TFFs), and flavanones/dihydroflavonols (TFDs). The small-intestinal phase was performed by two models: centrifugation and dialysis. The results of propolis-HP-β-CD extracts demonstrated high polyphenolic bioaccessibility during all three phases of digestion. The bioaccessibility of TPs, TFFs, and TFDs after the intestinal phase with centrifugation was from 0.02 to 8.86 times higher than in the dialysis phase, stressing that it is a better model than dialysis. Our findings show that the inclusion complex of propolis with HP-β-CD enables the good solubility of polyphenol groups.

Wild animals represent a constant source of Trichinella spp. infections for domestic animals and humans. To date, four species of Trichinella have been isolated in wild boar populations in Europe: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in addition to several mixed infection types and one hybrid formation between T. britovi and T. spiralis. Meanwhile, insufficiently thermally processed wild boar meat has been reported to be a source of trichinellosis in humans in several European countries. In Croatia, there have been no reported or proven cases of trichinellosis caused by wild boar meat consumption. The aim of this study was to obtain data on the prevalence of Trichinella species present in Croatia and to anticipated the potential risk of infection for humans in specific Croatian regions based on information obtained over an eight-year surveillance period. A veterinary inspection of wild boar carcasses for Trichinella larvae in Croatia has been mandatory since 1989, and the artificial digestion method was introduced as a compulsory test for wild boar samples in 2008. Based on the official data submitted to the Ministry of Agriculture, Directorate of Veterinary Services, in the period 2010–2017, 303 of 183,184 (0.17%) wild boar meat samples tested positive for Trichinella spp. Infected wild boar were found in 18 of 21 counties. Of these positive samples, 85 were submitted by the authorised veterinary inspectors to the National Reference Laboratory for further examination. The intensity of infection in muscle samples was 0.04–152.66 (mean: 23,37) larvae per gram, and Trichinella species were identified as T. spiralis, T. britovi, T. pseudospiralis and T. spiralis + T. britovi. Genetic analysis of T. pseudospiralis isolates demonstrated their belonging to the Palaearctic population.

Noble pen shells (Pinna nobilis) along the Eastern Adriatic coast were affected by mass mortalities similarly to the populations across the Mediterranean basin. Samples of live animals and organs originating from sites on Mljet Island on the south and the Istrian peninsula on the north of the Croatian Adriatic coast were analyzed using histology and molecular techniques to detect the presence of the previously described Haplosporidium pinnae and Mycobacterium spp. as possible causes of these mortalities. To obtain more information on the pattern of the spread of the mortalities, a study was undertaken in Mljet National Park, an area with a dense population of noble pen shells. The results of the diagnostic analysis and the velocity of the spread of the mortalities showed a significant correlation between increases in water temperature and the onset of mortality. Moderate to heavy lesions of the digestive glands were observed in specimens infected with H. pinnae. A phylogenetic analysis of the detected Haplosporidium pinnae showed an identity of 99.7 to 99.8% with isolates from other Mediterranean areas, while isolated Mycobacterium spp. showed a higher heterogeneity among isolates across the Mediterranean. The presence of Mycobacterium spp. in clinically healthy animals a few months before the onset of mortality imposes the need for further clarification of its role in mortality events.