Bovine mastitis (BM) has resulted in enormous economic loss in the dairy industry and coagulase-negative staphylococci (CNS) have caused subclinical BM. Although VITEK 2 GP ID card (VITEK 2) has been used for CNS identification, the probability of identification varies. The rpoB sequence typing (RSTing) method has been used for molecular diagnosis and epidemiology of bacterial infections. In this study, we undertook RSTing of CNS and compared the results with those of VITEK2 and 16S rRNA gene sequencing. As compared VITEK2, the molecular-based methods were more reliable for species identification; moreover, RSTing provided more molecular epidemiological information than that from 16S rRNA gene sequencing.
Animals ; Bacterial Infections ; Cattle ; Diagnosis ; Epidemiology ; Female ; Genes, rRNA ; Mastitis, Bovine ; Methods

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.
Agriculture ; Animals ; Cattle ; Computer Simulation ; Dairy Products ; Databases, Nucleic Acid ; Drug Resistance, Microbial ; Female ; Foodborne Diseases ; Humans ; Mastitis, Bovine ; Methods ; Molecular Epidemiology ; Prophages ; Staphylococcus aureus ; Staphylococcus

Objective: To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows. Methods: The milk sample was collected from a cow with mastitis, which was pretreated with 4% NaOH and inoculated with L-J medium for Mycobacterium isolation. The positive cultures were initially identified by acid-fast staining and multi-loci PCR, then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA, hsp65, ITS and SodA genes. The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay. Results: Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis, which were identified as non-tuberculosis mycobacterium by multi-loci PCR, and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis. The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics, including rifampicin and isoniazid, but they were sensitive to amikacin, moxifloxacin, levofloxacin, ethambutol, streptomycin, tobramycin, ciprofloxacin and linezolid. Conclusions:Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique. The results of the study can be used as reference for the prevention and control the infection in cows.
Animals ; Anti-Bacterial Agents/pharmacology* ; Antitubercular Agents/pharmacology* ; Cattle ; Drug Resistance, Bacterial ; Female ; Humans ; Mastitis, Bovine/microbiology* ; Microbial Sensitivity Tests ; Milk/microbiology* ; Mycobacterium/isolation & purification* ; Mycobacterium Infections/veterinary* ; Mycobacterium tuberculosis/drug effects* ; Nontuberculous Mycobacteria/isolation & purification* ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics*

OBJECTIVEIn this study, milk from a cow with mastitis was analyzed to determine the presence of mycobacterial infection. Milk quality and security problems pertaining to the safe consumption of dairy products were also discussed in this study.

METHODSMilk was preprocessed with 4% NaOH. Then, mycobacteria were isolated from the milk sample on L-J medium. The isolate was identified using multiple loci Polymerase Chain Reaction (PCR) and multi-locus sequence analysis with 16S rRNA, sodA, hsp65, and ITS genes. The drug sensitivity of the isolate to 27 antibiotics was tested through alamar blue assay.

RESULTSSmooth, moist, pale yellow colonies appeared on the L-J medium within a week after inoculation. Based on the results of multiple loci PCR analysis, the isolate was preliminarily identified as non-tuberculous mycobacteria. The 16S rRNA, SodA, hsp65, and ITS gene sequences of the isolate exhibited 99%, 99%, 99%, and 100% similarities, respectively, with those of the published reference strains of Mycobacterium elephantis (M. elephantis). The drug sensitivity results showed that the strain is resistant to isoniazid, p-aminosalicylic acid, and trimesulf but is sensitive to ofloxacin, rifampicin, amikacin, capreomycin, moxifloxacin, kanamycin, levofloxacin, cycloserine, ethambutol, streptomycin, tobramycin, rifabutin, ciprofloxacin, linezolid, cefoxitin, clarithromycin, and minocycline.

CONCLUSIONTo the best of our knowledge, this study is initially to report the isolation of M. elephantis from the milk of a cow with mastitis in China.

Animals ; Anti-Bacterial Agents ; pharmacology ; Cattle ; China ; Drug Resistance, Bacterial ; Female ; Mastitis, Bovine ; epidemiology ; microbiology ; Milk ; microbiology ; Mycobacterium ; drug effects ; genetics ; isolation & purification ; Mycobacterium Infections ; epidemiology ; microbiology ; veterinary ; Phylogeny ; Polymerase Chain Reaction

Bovine mastitis is an important microbial disease in the dairy industry. We investigated the frequencies of bacterial pathogens in 62 farms and pathogen antibiotic resistance from mastitis samples (n = 748). We tested the antimicrobial activity of chicken and duck egg white and lysozyme purified from chicken egg white. Moreover, we compared the microbiomes of normal and mastitic raw milk obtained by 16S rRNA gene pyrosequencing and culture methods. The results showed that the frequencies of Gram-positive pathogens (Enterococcus faecalis 37% and Staphylococcus aureus 36%) were higher than that of a Gram-negative pathogen (Escherichia coli 15%). Resistance frequencies to ampicillin and norfloxacin were lowest in Staphylococcus aureus (21%), Enterococcus faecalis (23%), and Escherichia coli (33%), and the antimicrobial activity of chicken egg white was higher than those of lysozyme and duck egg white. Pyrosequencing results revealed clear differences between the microbiomes of mastitic and normal raw milk samples and revealed a slightly similar, but clearly different, composition of pathogens compared to that from the culture method. Thus, pyrosequencing may be useful for elucidating changes in microbiomes during mastitis progression and treatment. A chicken egg white and antibiotic combination may help with mastitis treatment; however, further studies are needed.
Agriculture ; Ampicillin ; Animals ; Cattle ; Chickens ; Drug Resistance, Microbial ; Ducks ; Egg White* ; Enterococcus faecalis ; Escherichia coli ; Female ; Genes, rRNA* ; Mastitis ; Mastitis, Bovine* ; Methods ; Microbiota* ; Milk ; Muramidase ; Norfloxacin ; Ovum* ; Staphylococcus aureus

Oregano (Origanum vulgare) is an aromatic herb belonging to the Lamiaceae family. This study evaluated the therapeutic effect of oregano essential oil (OEO) on clinical bovine mastitis caused by Staphylococcus (S.) aureus and/or Escherichia (E.) coli. Thirty-two quarters from 18 lactating cows with subclinical mastitis were selected from a dairy farm and were divided into 4 groups (8 quarters/group): negative control (CON), positive control (GEN) and two OEO-treated groups (OEO-1 and OEO-2). Each group was intramammarily treated with saline, 10 g/tube of gentamicin ointment, and single and double doses of 0.9 mL OEO ointment twice a day for three days, respectively. After the treatments, physical udder conditions were greatly improved in GEN and OEO groups. In CON group, somatic cell counts (SCCs), number of white blood cells (WBC) and bacteria were increased. However, in OEO groups, SCCs and number of WBC were significantly decreased and S. aureus and E. coli were not detected in milk as compared with those before the treatments as well as GEN group. These results suggest that OEO may be a useful alternative to antibiotics for the control of subclinical bovine mastitis caused by S. aureus and/or E. coli.
Animals ; Anti-Bacterial Agents ; Bacteria ; Cattle ; Cell Count ; Escherichia coli* ; Escherichia* ; Female ; Gentamicins ; Humans ; Lamiaceae ; Leukocytes ; Mammary Glands, Animal ; Mastitis ; Mastitis, Bovine* ; Milk ; Origanum* ; Staphylococcus aureus* ; Staphylococcus*

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and alpha-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.
Animals ; Cattle ; Electrophoresis, Gel, Two-Dimensional/veterinary ; Escherichia coli/*physiology ; Escherichia coli Infections/genetics/immunology/microbiology/*veterinary ; Female ; Mammary Glands, Animal/*immunology/pathology ; Mastitis, Bovine/*genetics/immunology/microbiology ; Proteome/*genetics/metabolism ; *Proteomics

OBJECTIVETo investigate antimicrobial resistance profiles and genetic diversity of staphylococcus aureus isolated from lactating cows of 5 provinces in China, 2013.

METHODSA total of 680 samples were collected from 15 herds (12 farms, 3 artels) in 5 provinces of China in 2013, including swabs of extramammary sites (bovine teat skin and milking machine liners) and quarter milk samples from lactating cows diagnosed with subclinical mastitis. The antimicrobial resistance of the isolates were tested by broth microdilution method and the genotypes were determined by PFGE (pulsed-field gel electrophoresis) method.

RESULTSA total of 111 isolates were isolated and identified as staphylococcus aureus. Resistance to penicillin (90.1% (100/111)), erythromycin (48.6% (54/111)), ciprofloxacin (36.9% (41/111)), clindamycin (27.9% (31/111)), gentamycin (18.9% (21/111)), chloramphenicol (9.0% (10/111)), tetracycline (7.2% (8/111)) of these strains were observed. All isolates were sensitive to oxacillin, vancomycin and selectrin. 92.8% (103/111) staphylococcus aureus isolates were resistant to at least one antimicrobial. 38.7% (43/111) strains were multi-drug resistant isolates. The resistance rate of isolates in artels (100% (48/48)) was higher than it in farms (87% (55/63)) and the difference was statistically significant (χ(2) = 4.80, P < 0.05). The multi-resistance rate of isolates in artels (54% (26/48)) was higher than it in farms (27% (17/63)) and the difference was also statistically significant (χ(2) = 8.48, P < 0.05). The 111 strains were clustered into 8 types, 6 out of which were consisted of 98% isolates (109/111), and were prevalent in 2 to 9 herds. Every herd had 1 to 4 types, and tend to be comprised by one major type. Most swab isolates were indistinguishable from isolates infecting the mammary gland. There were no relationship between antimicrobial resistance profiles and genotypes of these isolates.

CONCLUSIONThe drug resistance of staphylococcus aureus isolates associated with lactating cows of 5 provinces in China is serious, especially the isolates collected from artels. A few specialized clones were responsible for most of the cases of bovine mastitis in a single herd and some clones might have a broad geographic distribution.

Animals ; Anti-Bacterial Agents ; Cattle ; China ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Female ; Genetic Variation ; Genotype ; Humans ; Lactation ; Mastitis, Bovine ; Microbial Sensitivity Tests ; Milk ; Staphylococcal Infections ; epidemiology ; Staphylococcus aureus ; genetics

The expression profiles of inflammatory cytokines viz. interleukins (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon-gamma and tumor necrosis factor-alpha in response to subclinical mastitis in indigenous cattle breed Kankrej (n = 6), Gir (Bos indicus) (n = 12) and crossbred (Bos taurus x Bos indicus) (n = 7) were investigated using quantitative real time PCR. Significant correlation (p < 0.05) was observed between total bacterial load and somatic cell count (SCC) in all three breeds of cattle. All the cytokines were observed to be up-regulated compared to cows with healthy quarters, however, level of their expression varied among three breeds of cattle. In Kankrej most cytokines were found to be transcribed to higher levels than in other two breeds; the milk had higher load of bacteria but not so high SCC, implying that Kankrej has a higher inherent resistance against mastitis. The results of present study indicated that mammary glands of crossbred cattle are more sensitive to bacterial infection than indigenous breed of cattle as they elicit immune response at lower bacterial load and result into higher SCC. Research on identification of factors responsible for differentially expressed cytokines profiles and use of cytokines as immunomodulatory tools can pave way for formulating control strategies against bovine mastitis.
Animals ; Bacteria ; Bacterial Infections ; Bacterial Load ; Cattle ; Cell Count ; Cytokines ; Female ; Granulocytes ; Interferon-gamma ; Interferons ; Interleukin-12 ; Interleukin-8 ; Interleukins ; Mammary Glands, Human ; Mastitis ; Mastitis, Bovine ; Milk ; Real-Time Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha

Changes in udder health and antibiotic resistance of mastitis pathogens isolated from dairies upon conversion from conventional to organic management over a 3-year period was studied. Coagulase-negative staphylococci (CNS) were the most prevalent mastitis pathogens isolated. CNS were significantly less resistant to beta-lactam antibiotics when isolated from milk after the herd transitioned to organic management. Cessation of the use of antimicrobial therapies in dairies in combination with organic management could lead to a reduction in the antimicrobial resistance of mastitis pathogens.
Ampicillin/pharmacology ; Animals ; Anti-Bacterial Agents/pharmacology ; Cattle ; Cephalothin/pharmacology ; Cloxacillin/pharmacology ; Drug Resistance, Microbial ; Female ; Lactation ; Mastitis, Bovine/*microbiology ; Microbial Sensitivity Tests ; Organic Agriculture ; Penicillins/pharmacology ; Prevalence ; Staphylococcal Infections/microbiology/*veterinary ; Staphylococcus/*drug effects/*isolation & purification