Antimicrobial resistance of bacterial isolates from sheep and goat cheeses in eastern Slovakia.

The aim of this study was to determine prevalence of undesirable bacteria and their antimicrobial profile in samples obtained from a productive farm situated in border region Slanské vrchy (Slovakia), involved in keeping sheep and goats for the purpose of processing raw milk to special products (cheeses). Genus and species identification was carried out by PCR method and MALDI -TOF MS. Isolates thus identified were detected for antimicrobial resistance using the Agar Dilution Method. Bacteria of Staphylococcus spp. exhibited the highest resistance to penicillin (98% isolates). Isolates from the family Enterobacteriacae showed the highest resistance to azithromycin (90%). At the same time, in isolates of Enterococcus spp. we detected high resistance to linezolid (100%). Our investigation showed that all tested strains were resistant to more than one antibiotic used in this study.

Short communication: Antimicrobial potential of Lactobacillus plantarum strains isolated from Slovak raw sheep milk cheeses.

The objective of this study was to screen Lactobacillus plantarum strains isolated from the traditional Slovak raw sheep milk cheese for their inhibitory potential. Seventy-two strains were obtained from samples of raw sheep milk and raw sheep milk cheeses collected from April 2017 to September 2018, in 23 geographical areas of Eastern Slovakia, by inoculation of de Man, Rogosa, and Sharpe agar plates (Oxoid, Basingstoke, UK). Using both the genus- and species-specific PCR methods, 43 strains were identified as Lactobacillus spp., and 10 strains were confirmed as Lb. plantarum. First, the whole bacterial cultures of Lb. plantarum strains were tested by disc diffusion assay. All showed very good antibacterial activities against 6 selected foodborne pathogens, including Escherichia coli, Salmonella Enteritidis, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus. Then, cell-free neutralized supernatants and partially purified bacteriocins were prepared from the 4 Lb. plantarum strains that exhibited the best antibacterial potential, and they were tested the same way as the whole bacterial cultures. Seven of the 10 Lb. plantarum strains harbored the plnEF gene, 3 strains harbored the plnD gene, and 2 strains possessed both the plnA and plnC genes that encode the production of the respective plantaricins. The presence of both plnR and plnL genes was only detected in a single Lb. plantarum isolate. Based on the results of this study, 4 strains of Lb. plantarum appeared to be suitable candidates for further testing in the dairy manufacturing sector, particularly in the production of raw sheep milk products.

Quantity of selected probiotic cultures in semi-hard cheese with low-cooking curd during the maturation process.

The work studies the survival of added selected probiotic bacteria Lactobacillus acidophilus (S1), Lactobacillus casei (S2), and Lactobacillus plantarum96 (S3) in semi-hard cheese with low-cooking curd during the maturation process. Cheeses were made according to the standard procedure (Polyfood SI 050 device). Probiotic lactobacilli strains Lactobacillus acidophilus (S1), Lactobacillus casei (S2), and Lactobacillus plantarum96 (S3) used in this study were added into the milk before the renneting process. The manufactured cheeses were matured for 6 months at the temperature of 10 °C. Cheese samples were taken for pH and titratable acidity measurements, lactobacilli enumeration, and chemical analysis at 30, 60, 90, 120, 150 and 180 days of maturation. At the end of the experiment (180 days) the cheese samples were analyzed also for the amount of lactic acid and protein contents. Initial numbers of lactobacilli inoculated into the milk (10(8) CFU mL(-1)) decreased during the first 2 weeks of maturation and reached from 2.15 10(7) CFU g(-1) in S1 cheese to 4.32 10(7) CFU g(-1) in S3 cheese. The number of Lactobacillus acidophilus strain bacteria at the beginning of the maturation period was 2.47.10(7) CFU g(-1) and declined until day 120 of maturation to the number of 0.45 10(6) CFU g(-1). In the last month of the experiment day 180 the viable cell numbers started to rise up to the final number of 0.41 10(7) CFU g(-1). The numbers of Lactobacillus plantarum96 varied around 10(8) CFU g(-1) during the whole period of the experiment. According to our results it was detected that in all experimental cheeses, the used probiotic lactobacilli reached the values above 10(6) CFU g(-1). Thus the legislated and therapeutic minimum limits set for the products containing probiotic bacteria for human diet were fulfilled.

Liquid chromatographic determination of tylosin in mastitic cow's milk following therapy.

Liquid chromatographic analysis of milk samples from 6 cows treated with tylosin in a veterinary practice indicated that tylosin persisted in milk for more than 3 days after the final treatment. The concentration of tylosin was not below the stated maximum residue limit (0.05 mg/kg). The milk from 3 cows being treated for mastitis catarrhalis chronica contained tylosin residues for 3.5 days after the last withdrawal time (72 h). No residue was detected in the milk of any animal 6 days after cessation of therapy.

[Oxytetracycline in the milk of dairy cows with clinical signs of mastitis during the lactation period]. / Vylucovanie oxytetracyklínu mliekom dojníc s klinickými prejavmi mastitídy pocas laktacného obdobia.

The objective of this study was to determine the oxytetracycline residues in milk from cows with clinical mastitis dosed with two extra-label routes of oxytetracycline administration not only during antibiotic's treatment (5 days), but also six days after treatment by use of a liquid chromatography method of testing with a detection limit of 20 ppb. Both groups of animals were treated once daily for five milkings at 24-hour intervals following morning milkings. Composite milk samples (equal volumes of foremilk from each quarter) were collected during morning and afternoon milkings, mixed together (1:1), and stored until analyzed. Milk samples were analyzed just before the first treatment (0 hour) and ten times at 24-hour intervals. Residue studies in milk cows indicate that oxytetracycline passes into milk. Residues in milk were higher for the cows receiving oxytetracycline by intramammary route (Tab. I) than for the cows receiving oxytetracycline intramuscularly (Tab. II). The highest mean data were 195.68 mg/kg after intramammary infusion (Fig. 2) and 2.74 mg/kg after intramuscular injection (Fig. 3) on the 5th day of the treatment beginning. The analysis data showed that oxytetracycline persisted in milk for as long as two days after both treatments at the concentration 0.03 mg/kg versus 0.02 mg/kg, respectively. No residues were detected in milk of any animal from the 4th day of the cessation of the therapy (Fig. 1) as detected by the HPLC method.

[Biochemical aspects of the toxic effects of Supermethrin in a 140-day avian reproductive test]. / Biochemické aspekty toxického úcinku supermetrínu v podmienkach chronického 140-dnového aviárneho reprodukcného testu.

Chronic toxic effects of supermethrin on some biochemical parameters (AST, ALT, LDH, creatinine and total proteins) were investigated in 84 individuals of Japanese quail divided into four groups (control-K, experimental group I-P1, experimental group II-P2, experimental group III-P3) in the conditions of 140-day avian reproductive test. The three experimental groups received the tested substance at these doses: P1-10.7 mg/kg l.w./day, P2-21.4 mg/kg l.w./day, P3-35.7 mg/kg l.w./day. The results of observation of the enzyme activities AST and ALT show that only the AST activity (in the course of 140-day avian reproductive test) significantly increased to 1.225 mu kat/l in the females of experimental group P1, to 1.053 mu kat/l in P2 and to 1.014 mu kat/l in P3 against the control, in which the AST activity was 0.670 mu kat/l. The values of AST activity in the males were 1.143 mu kat/l in P1, 1.117 mu kat/l in P2 and 1.090 mu kat/l in P3 against the control 0.8395 mu kat/l. The investigation of variations in total LDH activity in Japanese quail after 140-day avian reproductive test has shown an increase in the LDH activity in the males (11.193 mu kat/l in P1, 11.269 mu kat/l in P2, 8.245 mu kat/l in P3 and 7.362 mu kat/l in K) as well as in the females (10.91 mu kat/l in P1, 12.023 mu kat/l in P2, 10.196 mu kat/l in P3 and 7.055 mu kat/l in K).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of staphylococcal enterotoxins in milk and milk products by three methods.

For food evaluation, the determination of the number of Staphylococcus aureus colonies is insufficient in the view of present scientific knowledge. The results, advantages and disadvantages of diagnostic methods are demonstrated on an example of three methods of detection of staphylococcal enterotoxins in milk and milk products. 133 strains were investigated with the method of biotyping of Staphylococcus aureus strains. Four of their strains were included in biotype A, seven strains of S. aureus were not included in any biotype and the other strains belonged in biotypes C and E. This method can be used as an auxiliary method for evaluation of foods containing S. aureus bacteria. The agar-gel precipitation method of enterotoxin detection in isolated strains of Staphylococcus aureus has just restricted valiability. When examining 96 strains of S. aureus with this method, strains which were producing staphylococcal enterotoxins were isolated 17 times. The main disadvantage of this method is a fact that the result concerning the isolated strains need not be identical with the result of enterotoxin detection in food. Direct assays of staphylococcal enterotoxins in milk and milk products using an enzymoimmunological method (ELISA test) seem to be most promising, mainly due to their high sensitivity (0.0001-0.001 micrograms.ml-1) and other advantages.

[Biochemical aspects of the toxic effects of Supermethrin and the histochemical activity of alkaline phosphatase, acid phosphatase and non-specific esterase in subchronic poisoning in sheep]. / Biochemické aspekty toxického úcinku supermetrínu a histochemická aktivita alkalickej fosfatázy, kyslej fosfatázy a nespecifickej esterázy v podmienkach subchronickej intoxikácie oviec.

Fifteen Slovak Merino sheep were included in the experiment. The animals weighing 21-28 kg were divided into three groups per five animals. In a six-week feeding experiment the animals of group I were given 50 mg supermethrin per kg live weight per day while those of group II received 200, and from week four of the experiment 300 mg supermethrin per kg live weight per day. During the experiment changes of aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), acetylcholine esterase (EC 3.1.1.7), urea und creatinine levels in blood serum were observed. Six weeks after supermethrin treatment the sheep were slaughtered and histochemical evaluation of alkaline phosphatase (EC 3.1.3.2), acid phosphatase (EC 3.1.3.1) and non-specific esterase (EC 3.1.1.1) was carried out in liver, kidney, duodenum, jejunum and ileum. In the course of the experiment changes of the enzymatic activities of aspartate aminotransferase observed in both experimental groups of sheep were similar to those seen in the control group of animals (Tab. I). As compared to the starting values, no significant changes in the activity of alanine aminotransferase were observed in group II of the experiment and in the controls. However, a significantly decreased alanine aminotransferase activity could be seen in the blood serum of sheep of group I (Tab. II). In both experimental groups of animals no significant changes in the acetylcholine esterase could be seen (Tab. III). As compared to the starting values, no significant changes were observed in creatinine levels of the control and the 1st experimental group of sheep (Tab. IV). In the sheep of the 2nd group a temporary significant decrease (p < 0.05) in creatinine levels was seen. The dynamics of urea levels was similar to starting values in all animals throughout the experiment Tab. V). In the control group of animals (Fig. 1) the high density of reaction product of alkaline phosphatase was determined in the microvilli of enterocytes of the small intestine. In the small intestine of the animals of both experimental groups, the activity of this enzyme was shown to be located in the same zone (Fig. 2). In all experimental animals in the parenchyma of the liver and kidney no significant changes could be observed. In both experimental and control animals the high activity of acid phosphatase was demonstrated to be located especially in the cytoplasma of enterocytes. The activity of non-specific esterase was located in the cytoplasma of enterocytes of the small intestine, in the intestinal crypts its activity was slight up to high.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical signs and mechanism of supermethrin intoxication in sheep.

Three Slovak Merino sheep, weighing 38, 40 and 41 kg, were given single doses of 1500, 2700 or 3000 mg supermethrin/kg body weight. Clinical signs of intoxication were observed, and after death or sacrifice free cyanide levels were determined in the rumen contents and liver. The sheep that received 3000 mg supermethrin/kg had 7.2 and 0.58 mg cyanide/kg in the rumen contents and liver, respectively; the sheep that received 2700 mg supermethrin/kg had 5.8 and 0.52 mg cyanide/kg in the rumen contents and liver, respectively; whereas the sheep given 1500 mg supermethrin/kg had no free cyanide detected in the rumen contents or liver.