Virulence Factor Genes and Cytotoxicity of Streptococcus agalactiae Isolated from Bovine Mastitis in Poland.

Streptococcus agalactiae can produce a wide variety of virulence factors, including toxins and proteins which facilitate adhesion to and colonization and invasion of the host cells. There are few reports on the characteristics of field isolates from bovine mastitis in Poland. Thus, the aim of this study was to determine the occurrence of types of hemolysis on blood agar, virulence factor genes, and cytotoxicity of S. agalactiae isolates derived from cows with mastitis across Poland. The study included 68 isolates. Virulence genes were tested using standard PCR, and cytotoxicity was determined using methylthiazol tetrazolium (MTT) and lactate dehydrogenase (LDH) tests. Among the tested isolates, 89.7% were ß-hemolytic, 8.8% γ-hemolytic, and 1.5% alpha-hemolytic. The only genes detected in all isolates were the cfb, cspA, hylB, and sip genes. Cytotoxicity assessment based on the LDH test revealed that isolates were cytotoxic only to Vero cells. However, according to the results obtained from the MTT test, more than half of the isolates exhibited low cytotoxicity to both SK and Vero cells, whereas the other isolates showed moderate or no cytotoxicity to both cell lines. Our research confirms the prevalence of various virulence genes in S. agalactiae isolated from Polish dairy herds, which have previously been found in isolates recovered from human and animal infections. For the first time, the presence of bac- and scpB-positive isolates of S. agalactiae was determined in Polish dairy cattle, and the cytotoxicity of bovine isolates was assessed. IMPORTANCE We believe that this manuscript is one of the few reports on the characteristics of field S. agalactiae isolates derived from cases of bovine mastitis in cows in Poland in terms of the occurrence of virulence genes and cytotoxicity. For the first time, the presence of bac- and scpB-positive isolates of S. agalactiae was determined in Polish dairy cattle, and the cytotoxicity of bovine isolates was assessed.

The In Vitro Efficacy of Essential Oils and Antifungal Drugs Against Prototheca zopfii.

BACKGROUND: The algae of the genus Prototheca are environmental pathogens whose main reservoir is the habitat of cows. They can cause protothecosis in domestic and wild animals, as well as human beings, with the main etiological agents being Prototheca zopfii in animals and Prototheca wickerhamii in humans. AIM: The aim of the study was to evaluate the in vitro activity of selected essential oils and antifungal antibiotics against P. zopfii isolates. MATERIALS AND METHODS: The material consisted of nine P. zopfii strains isolated from the milk of cows suffering from mastitis. Eight essential oils produced by POLLENA-AROMA, Poland, and nine antifungal agents were tested. The effects of essential oils on P. zopfii were evaluated by microdilution with liquid Sabouraud dextrose broth, and susceptibility to antifungal agents was tested using the disk-diffusion method. RESULTS: All used essential oils inhibited the activity of P. zopfii isolates, with MIC values ranging from 0.2 to 10.5 µl/ml. Cinnamon, clove, and thyme demonstrated the highest activity against the tested P. zopfii strains at concentrations ranging from 0.6 to 1.0 µl/ml. Of the antifungal agents, the tested strains were the most sensitive to nystatin (100 %). CONCLUSIONS: The tested essential oils can be used to complement protothecosis therapy in animals and human beings.

Genotypic characterization and evaluation of an antibiotic resistance of Trueperella pyogenes (Arcanobacterium pyogenes) isolated from milk of dairy cows with clinical mastitis.

Trueperella pyogenes, recently reclassified from the genus Arcanobacterium, is considered the causative agent of acute suppurative mastitis called summer mastitis. T. pyogenes produces a variety of known and putative virulence factors that include pyolysin and factors promoting adhesion to host cells. The objective of this study was to report the presence of virulence genes in T. pyogenes isolates that were identified as etiological agents of clinical mastitis in cows, as well as to determine antimicrobial resistance and distribution of selected determinants that can be associated with phenotypic resistance among these isolates. The presence of genes (plo, nanH, nanP, cbpA, fimA, fimC, fimE, fimG, tet(W), erm(X), erm(B)) was examined by conventional PCRs. Resistance to 10 antimicrobial agents was determined by the broth microdilution method. Among T. pyogenes isolates of bovine mastitis origin the genes encoding all virulence factors occurred. Besides pyolysin gene plo, the fimA was the only gene detected in all isolates, whereas other virulence factor genes were found with different frequencies. Phenotypic antimicrobial resistance was observed to tetracycline (85.5% isolates) and erythromycin (9.1%). Isolates non-susceptible to erythromycin simultaneously exhibited increased MIC of pirlimycin. Beta-lactams were active against isolates. We found the correlation between the presence of tetracycline and macrolide resistance genes and corresponding resistance phenotype. Genotypic characterization of a large number of T. pyogenes isolates from different herds performed in this study may be useful in explanation, which virulence factors play a significant role in the establishment of bovine mammary gland infection.

Multicentre Etest evaluation of in vitro activity of conventional antifungal drugs against European bovine mastitis Prototheca spp. isolates.

OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC90 of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.

Effect of different heat treatments and disinfectants on the survival of Prototheca zopfii.

Bovine mastitis caused by the yeast-like alga Prototheca zopfii represents a serious veterinary problem and may result in heavy economic losses to particular dairy farms. The purpose of this study was to evaluate the survival of 50 isolates of P. zopfii in milk subjected to different heat treatments and the survival of further 106 P. zopfii isolates after exposure to three classes of teat disinfectants: iodine (Dipal), quaternary ammonium compounds (Teat), and dodecylbenzenesulphonic acid (Blu-gard). Of the 50 isolates tested for thermal tolerance, 29 (58%) survived heat treatment at 62 °C for 30 s and 13 (26% of all isolates) of those survived after heat treatment at 72 °C for 15 s. None of the 106 isolates were able to withstand the in-use concentrations of the three disinfectants tested. The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions: 1:1,000 for Dipal (survival rate of 52.8-57.5%), 1:100 for Teat (88.7-90.6%), and 1:10 for Blu-gard (100%). No differences in the survival rates of P. zopfii were observed with respect to the duration of exposure to disinfectant. The results of this study support the previous findings that P. zopfii may resist high-temperature treatments, including that applied in the high-temperature, short-time (HTST) pasteurization process. The obtained data also demonstrate the efficacy of the three classes of teat disinfectants against P. zopfii, with the efficacy of iodine being most pronounced. The study emphasizes the necessity of using higher temperatures in the pasteurization of raw milk to kill the Prototheca algae, as well as the particular suitability of the iodine for the control procedures of protothecal mastitis.

Genotyping of bovine Prototheca mastitis isolates from Poland.

Bovine mastitis due to unicellular, achlorophyllous algae of the genus Prototheca is a serious and complex ailment that accounts for high economic losses in the dairy industry. Bovine protothecal mastitis has been almost exclusively associated with only one species Prototheca zopfii. Recently, based on the 18S rDNA sequence analysis, P. zopfii has been divided into three genotypes (1-3), of which P. zopfii genotype 3 has been given the status of a new species Prototheca blaschkeae sp. nov. The aim of this study was to investigate the genotypic composition of the population of P. zopfii bovine mastitis isolates from Poland, according to that threefold classification. Of the 44 isolates tested, 43 (98%) were identified as P. zopfii genotype 2. Only one isolate was assigned to the newly established P. blaschkeae species. These findings clearly show the predominance of the P. zopfii genotype 2 in the etiology of bovine mammary protothecosis in Poland, but also underline the involvement of P. blaschkeae in the disease. The study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates, collected from different regions of Poland.

Sensitivity to antibiotics of Arcanobacterium pyogenes and Escherichia coli from the uteri of cows with metritis/endometritis.

The aim of the study was to determine the in vitro sensitivity of 161 Arcanobacterium pyogenes strains and 99 Escherichia coli strains isolated from the uteri of 312 cows with metritis/endometritis. Animals with pathological discharges were clinically diagnosed per rectum and per vaginum, and then swabs from the lumen of the uterus were aseptically collected. Bacteriological examination of swabs was according to commonly accepted procedures with the additional use of API tests. Sensitivity to antibiotics was tested by the disk diffusion method and performed in Mueller-Hinton agar. Arc. pyogenes strains were the most sensitive to amoxicillin/clavulanic acid (Amc) (97.3%), bacitracin (96.7%), ceftiofur (95.8%) and cephapirin (77.5%). E. coli strains were the most sensitive to norfloxacin (98.1%), marbofloxacin (95.8%), gentamycin (88%), Amc (80.7%) and ceftiofur (73.1%). Arc. pyogenes and E. coli were most resistant to oxytetracycline (63.7% and 31%, respectively).