Application of Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry for Identification of Coagulase-negative Staphylococci Isolated from Milk of Cows with Subclinical Mastitis.

The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of coagulase-negative staphylococci (CNS) isolated from the milk of cows with subclinical mastitis. The study material consisted of 33 isolates of CNS, identified by the results of API Staph tests, obtained from the milk of cows with subclinical mastitis. Based on the spectra analyses, MALDI-TOF MS tests of 33 bacterial samples allowed identification of the microorganisms in 27 cases (81.8%). The most frequent cause of subclinical mastitis was found to be Staphylococcus sciuri (39%), while S. vitulinus was detected in 15% of the milk samples. The results obtained indicate that MALDI-TOF MS can be used for the identification of CNS isolated from bovine mastitis as a method supplementary to biochemical tests.

Factors responsible for subclinical mastitis in cows caused by Staphylococcus chromogenes and its susceptibility to antibiotics based on bap, fnbA, eno, mecA, tetK, and ermA genes.

The aim of this study was to recognize selected factors of virulence determining the adhesion of Staphylococcus chromogenes to cows' udder tissues in subclinical mastitis and to evaluate the susceptibility of this pathogen to antibiotics. The subjects of the study were 38 isolates of Staph. chromogenes from 335 samples of milk from cows with subclinical coagulase-negative staphylococci mastitis. Somatic cell count ranged between 216,000 and 568,000/mL of milk (average 356,000/mL of milk). We confirmed the ability to produce slime in 24 isolates (63.2%), and the ability to produce protease in 29 isolates (76.3%). In each slime-producing isolate, the bap gene was not found, and the fnbA and eno genes were not detected. In vitro tests showed that ceftiofur had the highest effectiveness against Staph. chromogenes (89.5% of susceptible isolates). Minimum inhibitory concentrations ranged from 0.06 to 2µg/mL for susceptible isolates. The minimum concentrations required to inhibit growth of 90 and 50% of the isolates for ceftiofur were at or below the cutoffs recommended by the Clinical and Laboratory Standards Institute (2 and 0.06µg/mL, respectively). A significant percentage of the isolates were susceptible to other ß-lactam antibiotics: amoxicillin with clavulanic acid (84.2%) and ampicillin (81.6%). The lowest effectiveness among ß-lactams was for penicillin (73.7% of susceptible isolates), and the minimum inhibitory concentration for penicillin ranged from <0.06 to 8µg/mL. None of the examined isolates had the mecA gene, but ß-lactamase was detected in 4 isolates (10.5%). Erythromycin and oxytetracycline exhibited the lowest activity against Staph. chromogenes (71.1 and 63.2% of susceptible isolates, respectively). The genes tetK (6 isolates) and ermA (1 isolate) were also detected.

Sperm quality and selected biochemical parameters of seminal fluid in dogs with benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) in dogs is most commonly associated with age and increasing concentrations of dihydrotesterone, a hormone that stimulates growth and secretion of the prostatic epithelial cells. During this process, the biochemical composition of prostatic secretion changes, which can affect the quality of semen and limit the ability of the sperm to contribute to fertilization. Therefore, the present study was conducted to examine possible correlation between BPH and biological quality of semen. The study was performed in 11 sexually mature dogs of various breeds. Animals were divided into two groups: healthy dogs (Group I; n = 5; mean age 4.32; SEM = 1.28) and dogs with BPH (Group II n = 6; mean age 6.16; SEM = 0.65). Semen and prostate secretions were collected and evaluated in this study. Standard semen examinations were conducted in the ejaculates collected; moreover, the extent of apoptosis and DNA defragmentation was determined. The selected biochemical parameters were determined in the prostate secretion. According to the examination results, there were no significant differences in standard semen parameters between the two groups of dogs. Nevertheless, morphological tests of semen in dogs with BPH demonstrated elevated percentages of primary defects in spermatozoa. A significant increase (P = 0.01) in DNA defragmentation of sperm was found in dogs with BPH. Moreover, changes in the biochemical composition of prostate secretion were demonstrated. In dogs with BPH, pH of prostate secretions was greater (P = 0.03), concentrations of cholesterol increased while concentrations of Zn and Cu decreased. The study findings reveal that BPH does not change semen quality in dogs.

Inflammatory cytokines and acute-phase proteins concentrations in the peripheral blood and uterus of cows that developed endometritis during early postpartum.

The aim of the study was to evaluate the level of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6]), anti-inflammatory cytokine (interleukin-10 [IL-10]), and acute-phase proteins (haptoglobin [Hp] and serum amyloid A [SAA]) in serum and uterine washings in cows that developed endometritis during the early postpartum period. The study was carried out on 40 cows. The experimental group consisted of 20 cows with subclinical endometritis and the control group of 20 cows without endometritis. Analyses in both groups of cows were carried out at 5, 22, and 40 days postpartum (DPP). Experimental material consisted of the blood serum and uterine washings. The levels of the following cytokines: TNF-α, IL-6, IL-10 and acute-phase proteins: Hp and SAA were determined using ELISA. Our study reported that the levels of TNF-α, IL-6, IL-10, Hp, and SAA at 22 DPP were higher in cows with subclinical endometritis (P < 0.001). The levels of TNF-α (P = 0.01), IL-6 and IL-10 (P = 0.001), and Hp (P < 0.001) at 40 DPP were higher in cows with subclinical endometritis compared to healthy cows. The level of IL-10 in uterine washings at 5 DPP was higher (P = 0.001), whereas of SAA was lower (P = 0.01) in cows with subclinical endometritis. At 22 DPP, the levels of IL-6, IL-10, and Hp were higher (P < 0.001) in cows with endometritis. At 40 DPP, the level of TNF-α was lower, whereas these of IL-10 and Hp were elevated (P < 0.001) in cows with endometritis compared to healthy cows. The results indicate that the evaluation of the levels of cytokines and Hp in serum, but primarily in uterine washings, can be an important diagnostic indicator in cows that developed subclinical endometritis. High levels of IL-10 in cows with subclinical endometritis may contribute to the weakening of local resistance mechanisms of the uterus and lead to the persistence of the inflammation in the postpartum period. The present study also shows that the simultaneous examination of selected parameters of antagonistic interactions allows for better assessment of the current state of local immunity in the uterus.

Production of slime by coagulase-negative staphylococci (CNS) isolated from clinical and subclinical mastitis in cows.

The aim of the study was to evaluate the slime-producing ability of coagulase-negative staphylococci (CNS) isolated from clinical and subclinical mastitis in cows. The study was carried out on 100 isolates of CNS obtained from milk of 86 cows from farms located in the Lublin region (Poland). Slime-producing ability was observed in over half of coagulase-negative staphylococci (54.0% of isolated CNS), including 19 isolates of methicillin-resistant staphylococci (95.5% of all MRCNS). Of 22 isolates of CNS responsible for the clinical form of mastitis, 20 isolates (90.9%) produced slime: S. xylosus (7 isolates), S. haemolyticus (6 isolates), S. chromogenes (4 isolates), and S. sciuri (3 isolates), including 9 isolates of MRCNS (45.0%). The remaining 34 isolates of CNS (43.6%) with the ability to produce this exopolysaccharide were isolated from the milk of cows with subclinical form of mastitis: S. xylosus (12 isolates), S. sciuri (9 isolates), S. chromogenes (6 isolates), S. haemolyticus (3 isolates), S. warneri (3 isolates) and S. saprophyticus (1 isolate), including 10 isolates of MRCNS (12.8%).

Trueperella pyogenes and Escherichia coli as an etiological factor of endometritis in cows and the susceptibility of these bacteria to selected antibiotics.

The aim of this study was to determine the percentage of participation of particular species of microorganisms, isolated from the uterus of cows with endometritis and from cows without inflammatory lesions of the uterus, in the same postpartum period. The aim of the study was also to examine how long after parturition non-treated endometritis persists. Moreover, antibiotic susceptibility tests were carried out of the bacterial isolates dominating in the uterus. Forty cows were included in the study: 20 cows with endometritis (experimental group) and 20 cows without any inflammatory condition of the uterus (control group). The material for cytological and bacteriological tests was collected on the 5th, 26th, 40th and 60th day after parturition, using an intrauterine brush adapted for cows. The total number of collected isolates was 149, including 120 isolates from the uterus of cows with endometritis and 29 isolates from the uterus of cows without endometritis. The following species of microorganisms were isolated from the material collected from cows with endometritis: T. pyogenes (49.2%), E.coli (22.5%), F. necrophorum (11.7%), Staphylococcus sp. (6.7%), B. melaninogenicus (5.8%), and Streptococcus sp. (4.1%). The participation percentage of particular species of bacteria in the material collected from the uterus of cows without endometritis was as follows: T. pyogenes (27.6%), E.coli (24.2%), Staphylococcus sp. (20.7%), Streptococcus sp. (20.7%), B. melaninogenicus (3.4%) and F. necrophorum (3.4%). The highest percentage of T. pyogenes isolates was susceptible to ceftiofur (89.6%); cefoperazone (85.1%) and amoxicillin combined with clavulanic acid (79.1%). E. coli isolates were most susceptible to amoxicillin combined with clavulanic acid (100%), cefoperazone (94.1%) and oxytetracycline (82.3%).

Coagulase-negative staphylococci (CNS) as an aetiological factor of mastitis in cows.

The aim of the present study was to determine the proportions of individual coagulase-negative Staphylococcus species in clinical and subclinical mastitis. The material consisted of 100 CNS isolates obtained from 223 milk samples collected from cows with clinical and subclinical mastitis. Coagulase-negative staphylococci constituted 44.8% of all isolated microorganisms. CNS were isolated from the mammary gland secretions of 86 cows from farms in the Lublin region (Poland). Clinical mastitis was found in 20 whereas subclinical mastitis in 66 study cows (23.3% and 76.7%, respectively). The symptoms of clinical mastitis were mild. The clinical forms of mastitis concerned mainly the first or second lactation. Subclinical mastitis was most commonly observed during the second lactation. Four CNS species (S. xylosus, S. chromogenes, S. haemolyticus and S. sciuri) were isolated from clinical and subclinical mastitis. S. xylosus was the commonest CNS species isolated from cows with clinical mastitis whereas S. chromogenes was the most prevalent one in subclinical mastitis cases. The three CNS species (S. warneri, S. hominis and S. saprophyticus) caused only subclinical mastitis.

Resistance to methicillin of coagulase-negative staphylococci (CNS) isolated from bovine mastitis.

The aim of this study was to determine the mechanisms of staphylococcal resistance to methicillin. CNS (n = 100 isolates) were prepared from the mammary inflammatory secretions of 86 cows from farms located in the Lublin region. Methicillin-resistant isolates constituted 20.0% of all CNS. Staphylococcus sciuri (n=8) and Staphylococcus xylosus (n=6) were most abundant, followed by Staphylococcus chromogenes (n=3), Staphylococcus haemolyticus (n=2) and Staphylococcus warned (n=1). The mecA gene was found in 50.0% of MRCNS (10.0% of all CNS isolates) belonging to two species: S. sciuri and S. xylosus. All mecA-positive isolates contained the protein of low affinity to penicillin (penicillin-binding protein 2a - PBP2a). The enzyme hydrolysing the beta-lactam ring in antibiotics was detected in 40.0% of MRCNS; 10.0% of MRCNS isolates were characterised by the presence of the mecA gene and ability to produce beta-lactamase. The remaining 20.0% of MRCNS isolates showing phenotypic resistance to methicillin were mecA gene-negative and were not able to produce beta-lactamase.

Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows.

The aim of the present study was to assess the haemolytic and proteolytic activity of coagulase-negative staphylococci (CNS) isolated from cows with mastitis. The study was conducted on 100 CNS strains: S. xylosus (n=28), S. chromogenes (n=26), S. haemolyticus (n=25), S. sciuri (n=14), S. warneri (n=4), S. hominis (n=2), S. saprophyticus (n=1); 22 CNS were isolated from cows with clinical mastitis and 78 from those with subclinical mastitis. The CNS studied showed the ability to produce only alpha-haemolysin and belonged to one strain - S. haemolyticus (21.0% of isolated CNS strains). Haemolysin-positive CNS were responsible for both clinical and subclinical mastitis (22.7% and 20.5%, respectively). The ability to produce protease was found in 31.0% of CNS belonging to two strains: S. chromogenes and S. sciuri. Protease-positive CNS were the etiological factor of both clinical and subclinical mastitis (31.8% and 30.8%, respectively). All S. xylosus, S. warneri, S. hominis, and S. saprophyticus strains were found protease-negative and haemolysin-negative, irrespective of whether they caused clinical or subclinical mastitis in cows.

Antibiotic susceptibility of methicillin-resistant and methicillin-susceptible coagulase-negative staphylococci isolated from bovine mastitis.

The aim of the present study was to evaluate the antibiotic susceptibility of methicillin-susceptible (MS) and methicillin-resistant (MR) coagulase-negative Staphylococcus (CNS) strains isolated from milk of cows with mastitis. The study was conducted on 100 CNS strains (20 MRCNS and 80 MSCNS) isolated from milk samples of 86 cows from the Lublin (Poland) region farms. Antibiotic susceptibility of microorganisms was evaluated using the disc-diffusion method on the Mueller-Hinton agar according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). The highest efficacy against MSCNS was demonstrated for cephalosporin antibiotics, i.e. cefacetril (91.3%), ceftiofur (67.5%), cefoperazone (66.3%) and cephalexin (60.0% of susceptible MSCNS strains). Moreover, a high percentage of vancomycin-susceptible strains was demonstrated (83.8%). The activity of combination of amoxicillin with clavulanic acid and gentamicin was found weaker (63.8% and 61.3% of susceptible strains, respectively). About 50.0% of MSCNS were susceptible to erythromycin, enrofloxacine and amoxicillin. A large proportion of CNS was resistant to neomycin, penicillin, tetracycline, streptomycin, lincomycin and ampicillin (28.8%, 30.0%, 31.3%, 31.3%, 33.8% and 33.8% of susceptible strains, respectively). The highest percentage of MRCNS was susceptible to vancomycin (75.0%), erythromycin (65.0%) and streptomycin (50.0%). Their susceptibility to enrofloxacine (35.0%) as well as gentamicin and tetracycline (30.0%) was markedly lower. The lowest activity was found for lincomycin and neomycin (20.0% of susceptible MRCNS strains, each).

Enzymatic activity of yeasts isolated from the inflamed mammary secretion in dairy cows.

The aim of the study was to evaluate enzymatic activities of yeasts isolated from inflammatory mammary secretion. The yeasts isolated from cows with clinical and sub-clinical mastitis (134 strains) included: Candida krusei (62 strains), Candida kefyr (48 strains), Candida lusitaniae (17 strains) and Candida famata (7 strains). The API ZYM system was used containing substrates to assess 19 hydrolytic enzymes. Substantial differences in the number and activity of hydrolyses were demonstrated in individual species. In Candida krusei, acid phosphatase showed the highest activity (4.36 points), in Candida kefyr and Candida lusitaniae--leucine arylamidase (4.93 and 4.25 points, respectively), in Candida famata--alpha-glucosidase (4.75 points). No activity of trypsin, chymotrypsin, alpha-galactosidase, beta-glucuronidase, alpha-mannosidase or alpha-fucosidase was observed in any of the yeasts examined.

Antifungal susceptibility of yeasts isolated from secrection of inflamed mammary glands in cows.

Antifungal susceptibility of 150 strains of yeasts isolated from the infected mammary secretion of cows was determined. Their susceptibility to amphoterycin B, nystatin, flucytosine, tioconazole, ketoconazole, miconazole, fluconazole and clotrimazole was evaluated using the disc diffusion method on Yeast Nitrogen Base (YNB) and filter paper discs. The most active antifungal agents in vitro were found to be: tioconazole--96.7%, nystatin--95.4%, amphoterycin--94.0%, and flucytosine--92.7% of susceptible strains; the least active were fluconazole and clotrimazole--39.3% and 60.0% of susceptible strains, respectively. The individual species of yeasts were characterized by varied susceptibility to individual therapeutics.

The dynamics of changes in erythrocyte glutathione peroxidase activity and serum selenium content during the periparturient period in sows.

The aim of the present study was the description of the dynamics in erythrocyte GSH-Px activity and serum selenium content changes in sows during 14 days before and 14 days after parturition. The experiment was performed on 36 gilts of 1-3 years old. All animals came from the same farm with a closed production system and were fed the same diet of unknown selenium content. Blood was collected 8 times: 13-14 days before parturition, 6-7 days before parturition, 48-72 hours before parturition, and 12-24 hours before parturition, as well as 12-24 hours after parturition, 48-72 hours after parturition, 6-7 days after parturition and 13-14 days after parturition, always after feeding. Blood was centrifuged to obtain serum and erythrocytes were hemolysed. Erythrocyte GSH-Px activity was determined by the Paglia and Valentine method and expressed in U per g Hb. Serum selenium content was determined by the atomic absorption method. GSH-Px activity 14 days before parturition reached 13.38 U/g Hb and significantly decreased around the 7th day before parturition to 11.26 U/g Hb. 72 hours before parturition the activity of the enzyme increased to 16.85 U/g Hb. Between the 72nd hour before and the 14th day after parturition GSH-Px activity remained constant. Serum selenium content did not differ significantly in the examined gilts and ranged between 88.9 ug/L and 121.87 ug/L. 24 hours before and 24 hours after parturition the selenium content reached 96.55 ug/L and 88.91 ug/L respectively.

C-reactive protein in monitoring and evaluation of effects of therapy of the MMA syndrome of sows.

The elevated concentration of CRP in sows at the early stage of the MMA reflected the inflammation in the reproductive tract and in the mammary gland. Monitoring the level of CRP during the first days after parturition may be used to diagnose early stages of the MMA and to start therapy. In the treated MMA sows the level of CRP steadily decreased, and by the end of the experiment it had returned to the normal level. In the non-treated MMA sows CRP persisted at a high level and at day 10 of the experiment it was 13 times higher than in the treated animals and 16 times higher than in controls. Measurements of serum CRP are valuable in monitoring the health status of sows after parturition and should be introduced as the diagnostic method of the MMA syndrome in sows. The determination of the concentration of serum CRP during the first two days after parturition should be used for diagnosis of the early stages of MMA syndrome in sows and to start therapy when clinical symptoms of MMA are still absent.

Pharmacokinetics of tylosin in broiler chickens.

Biological availability and pharmacokinetic properties of tylosin were determined in broiler chickens after oral (p.o.) and intravenous (i.v.) administration at a dose of 10 mg/kg. The calculated bioavailability--F%, by comparing AUC values--p.o. and AUC--i.v., ranged from 30%-34%. After intravenous injection tylosin was rapidly distributed in the organism, showing elimination half-life (t1/2 beta) values of 0.52 h and distribution volume (Vd) of 0.69 L/kg, at a clearance rate (Cl) of 5.30 +/- 0.59 ml/min/kg. After oral administration, tylosin has a similar distribution volume (Vd = 0.85 L/kg), while the elimination half-life t1/2 beta of 2.07 h was four times bigger than after i.v. administration at Cl = 4.40 +/- 0.27 ml/min/kg. The obtained value tmax = 1.5 h for tylosin after oral administration indicates that using this antibiotic with drinking water in broiler chickens is the method of choice. However, a relatively low value Cmax = 1.2 micrograms/ml after oral administration of tylosin shows that dosing of this antibiotic in broiler chickens should be higher than in other food producing animals.

[Effect of immunostimulating agents on the occurrence of puerperal diseases in swine]. / Einfluss von Immunität stimullerenden Präparaten auf das Vorkommen der Puerperalerkrankungen bei Schweinen.

Investigations were done on effects of levamisol, chlormetine and biomast Cu. 22 upon the periparturient disease piglets. It was proved that levamisol and chlormetine applied prophylactically in immunostimulative doses at the last period of pregnancy exerted a favourable influence on the health state of sows after delivery. The piglets after sows previously subjected to immunostimulation with levamisol and chlormetine showed higher body mass gains per day, higher body mass on the 28th day after delivery, smaller number of diarrhoeas and death rate. A number of the reared piglets per one sow was also bigger. Preventive application of biomast administered at the end of pregnancy period turned out to be little effective in prophylaxis of periparturient diseases in sows. However, a positive influence of the above mentioned immunostimulation was stated in reference to some health and breeding indices.

[Effect of post partum intra-uterine infusion of Infertin ST on the occurrence of post partum diseases and the fertility of sows and on the health and breeding rate of piglets]. / Einfluss einer intrauterinen post partum durchgeführten Infusion von Infertin ST auf das Vorkommen von Postpartalerkrankungen und die Fruchtbarkeit der Sauen und auf die Gesundheits- und Zuchtindizes der Ferkel.

Investigations were carried out on the effect of Infertin ST intra uterine infusion after delivery upon the occurrence of postparturient diseases as well as health and breeding indices. It was demonstrated that a preventive Infertin ST intra uterine infusion performed after the parturition completion resulted in a decrease of the rate of sows with a raised temperature and mastitis. The intra uterine administration of Infertin ST had a favourable influence on sow fertility. The piglets of the sows treated with Infertin ST showed higher body mass gains, higher body mass on the 28th day of life and a lesser rate of diarrhoeas.

[Effect of hysterectomy in swine on health status and various indicators of peripheral blood. I. Clinical observations]. / Wplyw amputacji macicy u swin na stan zdrowia i niektóre wskazniki krwi obwodowej. I. Obserwacje kliniczne.

Studies were carried out on 36 sows from 100-250 kg of body weight, divided into 5 groups. The sows of group I were given Stresnil and Vetbutal to examine the effect of the anesthetics used during operation on the health condition of the animals studied in the other groups. Laparotomy was performed in group II, amputation of unpregnant uterus in group III, amputation of pregnant uterus or several hours after delivery in group IV, and amputation of pathologically changed uterus by the intraabdominal method and of fallen out uterus after delivery by the extra-abdominal method in group V. It was found that laparotomy, amputation of unpregnant uterus and that several hours after delivery were not a serious danger to the life of pigs, but general disturbances occurring after the operation depended on the magnitude of the operational trauma and health condition before operation. The treatment of pathologically changed uterus by intraabdominal amputation gave positive results in 69.2% of cases. However, no positive results were obtained in the treatment of fallen out uterus after delivery by extraabdominal amputation.

[Effect of hysterectomy in swine on health status and various indicators of peripheral blood. II. Hematological studies]. / Wplyw amputacji macicy u swin na stan zdrowia i niektóre wskazniki krwi obwodowej. II. Badania hematologiczne.

The effect of uterus amputation in various physiological and pathological conditions on the morphological picture of the peripheral blood in pigs was studied. It was found that amputation of the uterus in sows caused a decrease in the level of haematocrit and haemoglobin, in the number of erythrocytes and an increase of leucocytes. In the leucocyte picture an increase of segmented and rod-shaped granulocytes and a decrease of lymphocytes, eosinophils and basophils were found. The greatest changes observed were in the group of sows which were amputated pregnant uterus, the uterus short after delivery and that pathologically changed. The return of most of the indices studied to physiological values depended on the magnitude of the operation trauma and health condition of the pigs before operation.

[Effect of hysterectomy in swine on health status and various indicators of peripheral blood. III. Serum electrolyte levels]. / Wplyw amputacji macicy u swin na stan zdrowia i niektóre wskazniki krwi obwodowej. III. Zachowanie sie elektrolitów w surowicy krwi.

The studies carried out on sows which were amputated the uterus in various physiological and pathological conditions showed that this operation caused a decrease in the content of sodium and chloride and an increase of magnesium in blood serum. The changes in the level of electrolytes were the highest in the groups of sows which were amputated unpregnant and pregnant uterus and that short after delivery.