Clinical Cases Of Bovine Mastitis Research Articles

Whole genome sequence-based analysis of Staphylococcus aureus isolated from bovine mastitis in Thuringia, Germany.

Bovine mastitis is a common disease of dairy cattle causing major economic losses due to reduced yield and poor quality of milk worldwide. The current investigation aimed to gain insight into the genetic diversity, antimicrobial resistance profiles and virulence associated factors of Staphylococcus (S.) aureus isolated from clinical bovine mastitis in dairy farms in Thuringia, Germany. Forty Staphylococcus aureus isolates collected from clinical bovine mastitis cases from 17 Thuringian dairy farms were phenotyped and genetically characterized using whole genome sequencing. Out of 40 S. aureus, 30 (75%) were confirmed as methicillin resistant isolates. The isolates showed elevated antimicrobial resistance against penicillin, tetracycline and oxacillin, i.e., 77.5, 77.5, and 75%, respectively. Lower resistance rates were found against moxifloxacin, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, i.e., 35, 35, 30, and 22.5%, respectively. While resistance against clindamycin and erythromycin was rarely found (5 and 2.5%, respectively). All isolates were susceptible to linezolid, teicoplanin, vancomycin, tigecycline, fosfomycin, fusidic acid and rifampicin. These isolates were further allocated into five different sequence types: ST398 (n = 31), ST1074 (n = 4), ST504 (n = 3), ST582 (CC15) (n = 1) and ST479 (n = 1). These isolates were also assigned to seven clusters with up to 100 SNP which has facilitated geographical mapping and epidemiological distribution in Thuringia. Strains belonging to ST398 were classified into clusters 1, 2, 3, 4 and 7. The isolates of ST504 were of cluster 5, those of ST1074 were belonging to cluster 6. Resistance genes blaZ, blaI and blaR associated with penicillin resistance were found in 32 (80%) strains, all except one were belonging to ST398. Methicillin resistance associated mecA was identified in 30 (96.8%) isolates of ST398. All tetracycline and erythromycin resistant isolates were of ST398, and all harbored both tetM and ermA. About 90.3% of tetracycline resistant isolates assigned to ST398 were also carrying tetK gene. The point mutations parC_S80F, gyrA_S84L and parC_S80Y in gyrA and parC associated with quinolone resistance were found in all phenotypically resistant isolates to ciprofloxacin and moxifloxacin (n = 14). Sixty-eight virulence genes were identified among isolates. Both lukD/E and lukM/F-PV-P83 were identified in 22.5% of isolates, all were non-ST398. In this study, ST398 had the highest potential to cause disease and had a massive prevalence in bovine mastitis cases. Five different sequence types and seven clusters were identified in the federal state of Thuringia. The circulation of some clusters in the same region over several years shows the persistence of cluster-associated infection despite the intensive veterinary care. On the other hand, some regions had different clusters at the same year or in different consecutive years. Different sequence types and associated different clusters of S. aureus were geographically widely distributed among dairy farms in Thuringia. The findings of this study show that various clusters have the potential to spread over a large geographical scale. The detection of LA-MRSA on dairy farms, which is known for cabapility to widely spread among different groups of animals, humans and their environment urges for the implementation of national wide strategic programs. The identification of CA-MRSA among the isolates such as ST398 poses a significant risk for the transmission of such strains between animals and humans on dairy farms.

Antimicrobial activity of essential oils against Staphylococcus aureus and Staphylococcus chromogenes isolated from bovine mastitis.

Staphylococcus aureus and Staphylococcus chromogenes are pathogens frequently detected in bovine mastitis. Treatment and prevention of this disease have been usually carried on with antimicrobials. However, the emergence of bacterial isolates with antimicrobial resistance has aroused interest in new therapeutic alternatives. Plant essential oils (EOs) have been largely studied as antibacterial treatments. In the present study, EOs from five plants were evaluated for their antibacterial activities against S. aureus and S. chromogenes. Bacterial isolates were obtained in a previous study of clinical cases of bovine mastitis. EOs from lemongrass, eucalyptus, lavender, peppermint, and thyme were obtained by hydrodistillation and their chemical compositions were evaluated by gas chromatography (GC). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated for all EOs. The results demonstrated that citral (40.9%), myrcene (24.7%), and geraniol (1.9%) were detected in lemongrass EO; 1,8-cineole (76.9%), α-pinene (8.2%), and ledene (5.1%) in eucalyptus EO; 1,8-cineole (45.2%), camphor (18.2%), and fenchone (14.6%) in lavender EO; L-menthol (38.5%), menthofuran (16.3%), and citronellal (10.6%) in peppermint EO; and thymol (44.2%), p-cymene (24.6%) and 1,8-cineole (9.9%) in thyme EO. More effective antibacterial activities were observed only with the use of lemongrass (MIC and MBC ranging from 0.39 to 3.12mg/mL and 0.39 to 6.35mg/mL, respectively) and thyme (MIC and MBC ranging from 0.39 to 1.56mg/mL and 0.39 to 3.12mg/mL, respectively). Peppermint, lavender and eucalyptus EOs did not show bactericidal activities. In conclusion, lemongrass and thyme EOs are promising antibacterial alternatives against Staphylococcus species associated with bovine mastitis.

Prevalence and Characterization of Serratia marcescens Isolated from Clinical Bovine Mastitis Cases in Ningxia Hui Autonomous Region of China.

This study aimed to investigate the prevalence and genetic characterization of Serratia marcescens isolates from clinical bovine mastitis in Ningxia Hui Autonomous Region of China. S. marcescens was identified by the polymerase-chain reaction of 16S rRNA gene and sequencing. Antimicrobial susceptibility was tested by the disk diffusion method. Genes of resistance and virulence were determined by the PCR. Overall, S. marcescens were confirmed from 32 of 2897 (1.1%) mastitis milk samples. These isolates showed high resistance to cefazolin (30/32, 93.8%) and chloramphenicol (28/32, 87.5%). A 12.5% (4/32) of the isolates displayed multidrug resistance (MDR). The most prevalent resistant genes found in S. marcescens were TEM (32/32, 100%) and CTX-M (24/32, 75.0%; CTX-M-15, 14/32, 43.8%; CTX-M-14, 8/32, 25.0%; CTX-M-65, 2/32, 6.3%) for extended-spectrum beta-lactamase, cmlA (28/32, 87.5%) and floR (16/32, 50.0%) for chloramphenicol resistance, SIM-1 (2/32, 6.3%) for carbapenemases, and sdeB (28/32, 87.5%), sdeY (26/32, 81.3%), sdeR (26/32, 81.3%) and sdeD (20/32, 62.5%) for efflux pumps. Moreover, all isolates carried virulence genes flhD, entB, and kpn, and most of them contained mrkD (30/32, 93.8%), ycfM (26/32, 81.3%), bsmB (26/32, 81.3%), pigP (26/32, 81.3%), kfu (24/32, 75.0%) and shlB (24/32, 75.0%). To our knowledge, this is the first report of genetic determinants for antimicrobial resistance and virulence in S. marcescens isolated from bovine mastitis cases in China. These findings are useful for developing strategies for prevention and treatment of bovine mastitis caused by S. marcescens in China.

The prevalence of coagulase-negative staphylococcus associated with bovine mastitis in China and its antimicrobial resistance rate: a meta-analysis.

To contribute to the treatment decision and optimize coagulase-negative staphylococcus (CNS) control programs, we conducted a meta-analysis to investigate the epidemiology and antimicrobial resistance rates of coagulase-negative staphylococcus associated with bovine mastitis in China. Three databases (PubMed, Google scholar and China National Knowledge Infrastructure database) were utilized to obtain relevant publications. A total of 18 publications were included in our research, and 3 of them included antimicrobial resistant (AMR) test. The pooled prevalence of coagulase-negative staphylococcus was 17.28%. Subgroup analysis revealed that the prevalence was higher in South China than in North China, was higher in 2011-2020 than in 2000-2010 and was higher in clinical bovine mastitis cases than in subclinical cases. The pooled AMR were most resistant to β-lactams, followed by tetracyclines, quinolones, nitrofurans, lincosamides, sulfonamides, amphenicol and aminoglycosides. The pooled AMR rate of coagulase-negative staphylococcus was lower in 2011-2020 than in 2000-2010. Although the prevalence of CNS showed an increasing trend over 20 years, the AMR rate showed a decreasing trend, and the clinical type of mastitis was the most frequent and the prevalence was highest in South China. Finally, CNS was most resistant to β-lactams amongst the eight groups of antimicrobial agents.

In Vitro Antibacterial Activity of Selected South African Plants against Drug-Resistant Staphylococci Isolated from Clinical Cases of Bovine Mastitis

Bovine mastitis in dairy livestock production is a serious economic problem causing milk production losses and increased management costs. Staphylococcus aureus and non-aureus staphylococcus (NAS) are important causes of bovine mastitis. Antimicrobial resistance can limit the control of mastitis pathogens. Antibacterial and cytotoxic activities of acetone and ethanol extracts of nine South African plants were determined against reference and drug-resistant staphylococci isolated from clinical cases of bovine mastitis. The nine plants, namely, Antidesma venosum, Elaeodendron croceum, Erythrina caffra, Indigofera frutescens, Pleurostylia capensis, Searsia lancea, Searsia leptodictya, Trichilia emetica, and Ziziphus mucronata, were chosen for research material. The antibacterial activity of extracts was determined using a serial microdilution method, while a tetrazolium-based assay was used to determine their cytotoxicity against Vero cells. The values of antibacterial minimum inhibitory concentration (MIC) obtained were promising, with MIC ranging between 0.01 and 1.41 mg/mL. Generally, acetone extracts of most plants had better activity than their ethanol counterparts. The S. aureus strains were more susceptible to all the extracts than the NAS strains. The plant extracts’ half-maximal toxicity (LC50) was higher than 0.02 mg/mL, which is the recommended cytotoxic cut-off concentration. The ethanol extract of E. caffra, which showed better MIC values than its acetone extract, had the highest mean selectivity index (SI) of 8.30. Among the plants tested, S. lancea exhibited the most potent antibacterial activity, suggesting its potential usefulness as a broad-spectrum antibacterial agent against staphylococci associated with bovine mastitis, at relatively non-cytotoxic concentrations. Therefore, it is recommended for further investigation.

Virulence factors, antimicrobial resistance and phylogeny of bovine mastitis-associated Streptococcus dysgalactiae.

We carried out a thorough genetic evaluation of Streptococcus dysgalactiae isolated from clinical bovine mastitis cases and performed a phylogenetic analysis to represent the evolutionary relationship between S. dysgalactiae sequences. A total of 35 S. dysgalactiae strains were isolated from cases of clinical mastitis identified at a large commercial dairy farm located near Ithaca, New York. Whole-genome sequencing identified twenty-six antibiotic resistance genes, four of which were acquired genes, in addition to fifty virulence genes. Multi-locus sequence typing detected three new sequence types (STs). We conclude that a high proportion of this microorganism carries multiple virulence determinants and resistance genes, and that this indicates its potential to cause mastitis. Eight different STs were identified, of which ST453 (n = 17) was the most prevalent and ST714, ST715, ST716 were novel STs.

High Biofilm-Forming Ability and Clonal Dissemination among Colistin-Resistant Escherichia coli Isolates Recovered from Cows with Mastitis, Diarrheic Calves, and Chickens with Colibacillosis in Tunisia

Escherichia coli (E. coli) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC. A total of 120 samples including samples of milk (n = 70) and feces (n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship. Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The blaTEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the blaCTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fimA gene (26/36, 72.2%), followed by aer (12/36, 33.3%), cnf1 (6/36, 16.6%), papC (4/36, 11.1%), and stx1 and stx2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia. The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Antimicrobial resistance and virulence traits of Enterococcus faecium isolated from clinical bovine mastitis in Ningxia, China

This study was conducted to determine the antimicrobial resistance and virulence traits of 32 Enterococcus faecium isolates from clinical bovine mastitis cases in Ningxia Province, China. In total, 32 E. faecium isolates were taken from subclinical bovine mastitis on the basis of morphological characterization and biochemical testing, and screened for antimicrobial susceptibility. The virulence genes of the isolates were studied using polymerase chain reaction (PCR). The disc diffusion assay revealed a high occurrence of resistance against tetracycline (78.1%) and erythromycin (68.8%) in the E. faecium isolates. However, all tested E. faecium were susceptible to linezolid and vancomycin. Moreover, all E. faecium isolates harbored the erythromycin-resistant genes ermA, ermB and ermC, as well as the tetracycline-resistant genes tetK, tetL and tetM. Furthermore, all E. faecium isolates carried more than 3 of the tested virulence genes. The presence of agg (100%), cpd (100%), efaA (100%), gelE (93.4%), and esp (75.0%) was found most frequently in all the tested isolates. These findings are useful for making appropriate antimicrobial choices and developing antivirulence therapies for subclinical bovine mastitis caused by E. faecium in Ningxia Province, China.

Emulsion-Based Postbiotic Formulation Is Comparable to Viable Cells in Eliciting a Localized Immune Response in Dairy Cows With Chronic Mastitis.

Bovine mastitis is a disease with a multi-etiological nature, defined as an infection and inflammation of the udder. Mastitis represents a significant ongoing concern in the dairy industry, leading to substantial losses in profits and revenue for farmers worldwide. The predominant causes of bovine mastitis include the pathogens Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and Escherichia coli. Antibiotic administration is currently the main treatment option for mastitis. However, there is a pressing need for alternative therapies to treat and prevent the disease, especially with the emergence of antibiotic-resistant, mastitis-causing pathogens, resulting in antibiotic treatment failure. One such example is live bio-therapeutics (also known as probiotics), such as Lactococcus lactis DPC3147. The efficacy of this live bio-therapeutic has been demonstrated in several previous trials by our group. The most recent of these trials showed that an emulsion-based formulation of this strain was as effective as a commercial antibiotic formulation in treating sub-clinical and clinical cases of bovine mastitis. Here, we report the results of a follow-up field trial, in which we sought to gain insight into the mechanism of action of such live bio-therapeutics, focussing on chronic mastitis cases. We treated 28 cows with chronic mastitis with two separate emulsion-based formulations containing either viable L. lactis DPC3147 cells (15 cows) or heat-killed L. lactis DPC3147 cells (13 cows). We then evaluated the efficacies of the two formulations (two treatment groups) in terms of stimulating a localized immune response (quantified by measuring IL-8 concentrations in milk collected from udders affected by mastitis) and efficacies in terms of cure rates (quantified by reductions in somatic cell counts and absence of pathogens). We demonstrate that the presence of heat-inactivated bacteria (a postbiotic) was as effective as the live bio-therapeutic in eliciting a localized immune response in cows with chronic mastitis. The response to heat-killed cells (postbiotic) reported herein could have beneficial implications for farmers with regard to prolonging the shelf life of such emulsion-based formulations containing heat-killed cells of L. lactis DPC3147 for curing cows with mastitis.

Training, development, and evaluation of on-farm culturing as a technical resource to reduce the use of antimicrobials in dairy herds in the Midwest Region of Santa Catarina State, Brazil

ABSTRACT: On-farm culturing is a technology booming in Brazil. It is based on the microbiological diagnosis of milk samples collected from clinical cases of bovine mastitis, on the farm where the animals are handled, quickly, simply, and at an affordable cost. With this resource, after 24 h of incubation, the isolated microorganisms are classified as gram positive or gram negative, helping to make decisions regarding the therapy of the animals. Considering the relevance of the Midwestern region of Santa Catarina State in the national dairy production scenario, the objectives of the study were: 1) to promote the technical training of the farmers in three dairy farms located in Treze Tílias-SC, 2) to assist in the implementation and evaluation of on-farm culturing as technological resource viewing the reduced use of antimicrobials in herds. In addition to the rapid acceptance by the farmers and their efficient training, with the use of the technology, there was a 45-50% reduction in the use of antimicrobials in dairy herds. This result demonstrated a significant effect on animal health, and determines an important cost reduction for farmers, as presented in detail in this manuscript.

Antimicrobial Resistance and Virulence Factor of Streptococcus dysgalactiae Isolated from Clinical Bovine Mastitis Cases in Northwest China.

ObjectiveStreptococcus dysgalactiae is a major pathogen in bovine mastitis. The purpose of this study was to survey the prevalence, antimicrobial resistance, as well as the spread of resistance and virulence-associated gene of S. dysgalactiae.MethodsA total of 60 S. dysgalactiae strains were obtained from 830 milk samples from Holstein cows with clinical mastitis. Antimicrobial resistance was examined by the disk diffusion method. Antimicrobial resistance and virulence genes were investigated by PCR, agarose gel electrophoresis and 16S rRNA gene sequencing.ResultsAll isolates were resistant to tetracycline and showed a high level of resistance to aminoglycoside antibiotics, where 81.67% of the strains were multi-resistant to these ten sorts of antibiotics. In addition, the most prevalent resistance gene in S. dysgalactiae was aphA-1 (98.33%), followed by blaTEM (96.67%), ermB (83.3%), aadA1/aadA2 (78.33%) and tetL (73.33%). Totally, seven virulence genes with 25 combination patterns were detected in these isolates, and each isolates harbored at least one virulence gene. 21.67% of the isolates carried three or more virulence genes, while one strain with seven virulence-related genes and belonged to cfb+lmb+eno+napr+bca+scpB+cyl.ConclusionThese findings indicate that S. dysgalactiae isolated from clinical bovine mastitis cases in Northwest China show a variety of molecular ecology and are highly resistant to antibiotics commonly used in dairy farms. This research will help investigators better understand the pathophysiology S. dysgalactiae in bovine mastitis and choose the appropriate antibiotics to treat mastitis.

N-acetyl-β-D-glucosaminidase biomarker quantification in milk using Ag-porous Si SERS platform for mastitis severity evaluation

Bovine mammary gland is susceptible to numerous bacterial infections resulting in inflammatory disease condition of bovine mastitis (BM) with a staggering economic impact on the dairy industry worldwide. Early BM detection is crucial for infection control within the dairy herd, which can be alleviated by antimicrobial therapy. N-acetyl-β-D-glucosaminidase (NAGase) is a prominent BM inflammatory biomarker secreted onto the blood circulation upon pathogenesis and then released into milk, capable of separating healthy quarters from subclinical and clinical BM cases. Herein, we report a sensitive, cost-effective and handy-to-use BM severity assay based on NAGase inherent content found in whole milk samples. Silver-coated porous Si (Ag-PSi) Fabry–Pérot interferometers were employed for quantifying the lysosomal activity in different milk qualities using surface-enhanced Raman spectroscopy (SERS). The optical response was proportional to the occurring pathogenesis induced by predominant bacteria found in dairy farms at different somatic cell levels. The optimized Ag-PSi SERS substrates were utilized for quantitative analysis of NAGase levels found at clinically relevant BM concentrations while presenting the detection limit of 0.27 μM min−1. The optical performances were associated with the conventional standardized approach in terms of recovery values (85–98%), thus presenting sufficient adaptability to complex matrix analysis as whole milk.

Bovine mastitis inflammatory assessment using silica coated ZnO-NPs induced fluorescence of NAGase biomarker assay

Bovine mastitis (BM) is the most common inflammatory disease in the dairy sector worldwide, originated from bacterial invasion onto the mammary gland. Early BM detection is crucial for identifying new pathogenic infections within the dairy herd, which can be alleviated by antimicrobial therapy. N-acetyl-β-D-glucosaminidase (NAGase) is a prominent BM inflammatory biomarker secreted onto the blood circulation upon pathogenesis and then released into milk, capable of separating healthy quarters from subclinical and clinical BM cases. Herein, we report on a sensitive differentiation assay of BM severity based on enhanced fluorescence emission of a conventional NAGase activity assay. The addition of silica-coated zinc oxide nanoparticles induces non-radiative energy transfer to the lysosomal reaction products, thus leading to enhanced fluorescence (above 3-fold). Various milk qualities within the entire inflammatory spectrum were evaluated by the modified fluorescence assay with respect to non-infected milk. The amplified emission values differentiate between two predominant BM causative pathogens (Streptococcus dysgalactiae and Escherichia coli) at various somatic cell counts. In general, the presented concept offers an efficient, simple, cost-effective fluorescence signal augmentation for mastitis identification, thus offering means to diagnose the severity of the associated disease.

Characterization of Staphylococcus aureus Isolates From Cases of Clinical Bovine Mastitis on Large-Scale Chinese Dairy Farms.

Bovine mastitis is a prevalent disease that causes serious economic problems globally in the dairy industry. Staphylococcus aureus is an important pathogen of bovine mastitis. This study was conducted to characterize S. aureus isolates from clinical bovine mastitis cases in large-scale dairy herds in China. S. aureus was isolated from 624 clinical mastitis cases and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 62 S. aureus isolates were obtained. Cluster analysis, genetic diversity, quantification of biofilm formation, antimicrobial resistance, and detection of virulence genes were performed on these isolates of S. aureus. Eight isolates harbored the mecA gene and were sensitive to oxacillin. MALDI-TOF MS cluster analysis revealed that the 62 isolates were divided into three major clusters (I, II, III) and eight main groups (A–H) at the distance level of 700. The agr II was the most prevalent (56.5%). The 62 S. aureus isolates were assigned to seven spa types. The most common spa type was t529(58.1%), followed by t2196 (14.5%), t518 (14.5%), t571(6.5%), t034 (3.2%), t2734 (1.6%), and t730 (1.6%). Five STs were identified from seven representative isolates as follows: ST630/CC8, ST97/CC97, ST50, ST398, and ST705. All isolates had the ability to form biofilm. Antimicrobial resistance was most frequently observed to ciprofloxacin (29%), followed by penicillin (24.2%), and streptomycin (9.6%). All isolates harbored the fnbA, clfB (100%), icaA, and icaD genes. This study provides the basis for the development of bovine mastitis prevention program on large-scale dairy farms.

Antibacterial Activities of Acetic Acid against Major and Minor Pathogens Isolated from Mastitis in Dairy Cows.

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening of their antibacterial activities by agar well diffusion method was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each acid were obtained using a microdilution method; each acid was diluted from stock solution and then were diluted with culture broth to reach concentrations ranging from 4 to 0.004% w/v. The results showed acetic acid had the highest zone of inhibition against all pathogens except Escherichia coli compared with lauric and caprylic acids. The MIC and MBC were lowest for acetic acid against both Gram-positive (except Staphylococcus chromogenes from the coagulase negative staphylococci (CNS) group) and Gram-negative pathogens, intermediate for lactic and caprylic acids and greatest for lauric acid. In conclusion, acetic acid had antimicrobial activities against most mastitis pathogens compared with other acids. Further studies are needed to optimize the formulation and concentration of acetic acid for teat-dipping agent in the future.

Detection of antimicrobial resistance and virulence-related genes in Streptococcus uberis and Streptococcus parauberis isolated from clinical bovine mastitis cases in northwestern China

The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance- and virulence-related gene patterns. Antimicrobial susceptibility was determined by the E-test. Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR. A total of 27 strains were obtained from 326 mastitis milk samples. Streptococcus parauberis isolates (n=11) showed high resistance to erythromycin (90.9%), followed by tetracycline (45.5%), chloramphenicol (36.4%) and clindamycin (27.3%). Streptococcus uberis isolates (n=16) were highly resistant to tetracycline (81.3%) and clindamycin (62.5%). Both species were susceptible to ampicillin. The most prevalent resistance gene in S. uberis was tetM (80.0%), followed by blaZ (62.5%) and ermB (62.5%). However, tetM, blaZ, and ermB genes were only found in 27.3, 45.5, and 27.3%, respectively, of S. parauberis. In addition, all of the isolates carried at least one selected virulence-related gene. The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2% of the strains. One S. uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern. More than 59.3% of analysed strains carried 4 to 7 virulence-related genes. Our findings demonstrated that S. parauberis and S. uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology, and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry. The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens, and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.

Isolation and Identification of Major Mastitis Causing Bacteria from Clinical Cases of Bovine Mastitis in Kashmir Valley

Background: Mastitis is one of the most economically important diseases of dairy animals causing heavy losses in terms of fall in milk production, decreased milk quality and additional costs for veterinary services. Mastitis is caused by a wide variety of microorganisms including bacteria, fungi, yeast and mycoplasma, of which bacteria are the most frequently isolated pathogens. Microbiological testing is necessary for identification of cause of mastitis and adaptation of control measures to prevent the disease. The aim of the presnt study was to isolate and identify major mastitis causing bacteria from clinical mastitic cases presented to Veterinary Clinical complex, FVSc. and AH, Shuhama Kashmir for treatment. Methods: A total of 200 bovine milk samples from CMT confirmed clinical mastitis cases were collected and cultured on Nutrient agar, Blood agar, Mac-Conkey agar, Eosine Methylene Blue (EMB) agar, Mannitol salt agar and Edwards media for bacterial isolation. The isolates were then subjected to biochemical tests for identification. Result: Upon microbiological testing and biochemical confirmation, the major mastitis causing bacteria isolated from clinical cases of Bovine mastitis were Staphylococcus Spp. showing highest prevalence of 46.4% followed by mixed infection with Staphylococcus and Streptococcus (20.8%), Streptococci Spp. (18.4%) and Escherichia coli (14.4%).

Associations between Streptococcus uberis strains from the animal environment and clinical bovine mastitis cases

Bovine clinical mastitis quarter foremilk samples were collected from 15 German dairy farms for the isolation of Streptococcus uberis strains. Samples were also collected from the 8 spots where Streptococcus uberis was most expected in the dairy environment to investigate the transmission behavior of Streptococcus uberis within the farm. The selected environmental spots for sampling were the inner surface of the milking liner, drinking troughs (on pasture and in the barn), exit area of milking parlor, bedding material from the lying area in the barn, passageway to pasture, lying area of soil or vegetation on pasture, and the barn area in front of the milking parlor. We performed pulsed-field gel electrophoresis on 237 Streptococcus uberis isolates to identify environmental strains that matched those from mastitis milk. The same strains were detected on the passageway to the pasture, milking parlor waiting area, in one of the liners, and a drinking trough. Streptococcus uberis strains showed high variability within farms and because identical strains (in mastitis milk and environment) were found in different environmental localizations, its transmission appears to be farm specific. Thus, to establish a farm-specific mastitis control strategy, the main environmental sources of Streptococcus uberis must be analyzed for matching strains. A molecular method such as pulsed-field gel electrophoresis is an important tool that can be used to obtain the necessary information.

Biofilm production and other virulence factors in Streptococcus spp. isolated from clinical cases of bovine mastitis in Poland

Mastitis is a common disease in dairy cattle. An important aetiological agent of this disease is bacteria of the genus Streptococcus; hence, exploring the mechanisms of virulence in these bacteria is an extremely important step for the development of effective prevention programmes. The purpose of our study was to determine the ability to produce biofilm and the occurrence of selected invasiveness factors among Streptococcus isolated from cattle with the clinical form of mastitis in northeastern Poland. Most of the isolates analyzed demonstrated an ability to produce biofilm (over 70 %). Virulence genes were searched for in S. agalactiae, S. uberis and S. dysgalactiae. For S. agalactiae, only four genes were confirmed: rib (33 %), cylE (78 %), bca (37 %), and cfb (100 %). The genes pavA, scpB, bac and lmb were not present in any of the tested strains. The dominant serotypes were Ia (n=8) and II (n=8), in addition to some strains that were not classified in any of the groups (n=6). Out of the eight selected genes for S. uberis only one was not found (lbp). Finally, two genes were chosen for S. dysgalactiae (eno and napr), and their presence was confirmed in 76 % and 86 % of the strains, respectively. The experiment showed that strains of Streptococcus spp. isolated from dairy cattle with clinical cases of mastitis in the northeastern part of Poland possess several invasiveness factors that can substantially affect the course of the disease, and this should be considered when developing targeted prevention programmes.

Prevalence of bovine mastitis pathogens and antibioresistance of Escherichia coli isolates

Nine hundred and fifty two milk samples from clinical bovine mastitis cases, originated from 269 farms in Northern Greece, were tested. Escherichia coli was isolated in 49.3% of samples. Furthermore, Staphylococcus spp, Corynebacterium spp, Streptococcus spp, Pseudomonas spp, Proteus spp, Klebsiella spp or a combination of the above bacteria were isolated. The bacteriological result from 4.3% of samples was negative. No correlation between clinical mastitis cases and seasonal variation was observed. Resistance of E. coli isolates to tetracycline was 82-96%, to enrofloxacin 10-30%, to gentamicin 58-80%, to cephalosporins 47-75%, to sulfamethoxazole-trimethoprime 46-81%, to ampicillin 71-92% and to neomycin 83-97%. The highest resistance rate and the appearance of multi-resistant isolates of E. coli (6%), to 8 antibacterials, were recorded during the last year of the survey.