Bovine Mastitis In China Research Articles

Occurrence of antimicrobial resistance and class 1 integrons in Staphylococcus aureus isolated from bovine mastitis in China.

Integrons are important genetic elements that allow easy acquisition and dissemination of antimicrobial resistance genes. Studies reporting occurrence of integrons in Staphylococcus aureus (S. aureus) isolated from bovine mastitis in large dairy farms across China are scarce. The aim of this study was to investigate the occurrence of class 1 integrons (intI1), antimicrobial resistance (AMR) and associated genes in S. aureus isolated from bovine mastitis and their associations. Minimum inhibitory concentrations (MICs) were determined to evaluate the AMR phenotypes, whereas PCR was carried out to assess the occurrence of AMR genes and intI1. In addition, index cluster analysis was used to estimate associations between AMR phenotype, genotype and intI1 in 103 isolates. Overall, 83% of S. aureus were intI1-positive and 5 types of gene cassettes were detected. Susceptibility against single antimicrobial agents ranged from 0% (erythromycin), 12% (ampicillin) and 16% (penicillin G) to 96% (gentamicin). Most isolates (64%) were intermediate-resistant against erythromycin, whereas resistance against ceftriaxone (22%), clindamycin (4%), cefotaxime (2%), tetracycline (1%) and ciprofloxacin (1%) were relatively uncommon. The predominant resistant gene was blaZ gene (n = 88, 85%) followed by tetD gene (n = 85, 83%). With an estimated prevalence of 12% of the mecA gene, methicillin-resistant S. aureus isolates had higher MIC50 and MIC90 for majority of antimicrobials than methicillin-susceptible S. aureus isolates. Presence of the ermC gene was associated with erythromycin resistance. Ampicillin, erythromycin and penicillin G resistance were associated with intI1. The data presented in our study indicated that class 1 integron-mediated resistance possibly plays an important role in dissemination of AMR in S. aureus isolated from bovine mastitis.

Prevalence and molecular characterization of multi-resistant Escherichia coli isolates from clinical bovine mastitis in China

Different antibiotics are used to treat mastitis in dairy cows that is caused by Escherichia coli (E. coli). Antimicrobial resistance in food-producing animals in China has been monitored since 2000. Surveillance data have shown that the prevalence of multiresistant E. coli in animals has increased significantly. This study aimed to investigate the occurrence and molecular characteristics of resistance determinants in E. coli strains (n = 105) obtained from lactating cows with clinical bovine mastitis (CBM) in China. A total of 220 cows with clinical mastitis, which has swollen mammary udder with reduced and red or gangrenous milk, were selected from 5000 cows. The results showed 94.3% of the isolates were recognized as multidrug resistant. The isolates (30.5%) were positive for the class I integrase gene along with seven gene cassettes that were accountable for resistance to trimethoprim resistance (dfrA17, dfr2d and dfrA1), aminoglycosides resistance (aadA1 and aadA5) and chloramphenicol resistance (catB3 and catB2), respectively. The bla TEM gene was present in all the isolates, and these carried the bla CTX gene. A double mutation in gyrA (i.e., Ser83Leu and Asp87Asn) was observed in all fluoroquinolone-resistant isolates. In total, nine fluoroquinolone-resistant E. coli isolates were identified with five different types of mutations in parC. In four (44.4%) isolates, Ser458Ala was present in parE, and in all nine (9/9) fluoroquinolone-resistant isolates, Pro385Ala was present in gyrB. Meanwhile, fluoroquinolone was observed as highly resistant, especially in isolates with gyrA and parC mutations. In summary, the findings of this research recognize the fluoroquinolone resistance mechanism and disclose integron prevalence and ESBLs in E. coli isolates from lactating cattle with CBM.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Antimicrobial resistance and virulence profiles of staphylococci isolated from clinical bovine mastitis.

Staphylococci, mainly including Staphylococcus aureus and coagulase-negative staphylococci (CNS), are one of the most common pathogens causing bovine mastitis worldwide. In this study, we investigated the antimicrobial resistance and virulence profiles of staphylococci from clinical bovine mastitis in Ningxia Hui Autonomous Region of China. Antimicrobial resistance was determined by disc diffusion combined with E-test method. Genes of antimicrobial resistance and virulence factors were determined by PCR. A total of 332 staphylococcal isolates were confirmed from 1,519 mastitic milk samples, including 172 S. aureus and 160 CNS isolates. Fifteen CNS species were identified, with S. chromogenes being the most frequent found (49.4%), followed by S. equorum (13.8%). Noticeably, 2 S. agnetis isolates were found among the CNS isolates. To our knowledge, this is the first report documenting the presence of S. agnetis from bovine mastitis in China. The S. aureus and CNS isolates showed high resistance against penicillin, followed by erythromycin and tetracycline. Multidrug resistance was found in 11.6 and 16.3% of the S. aureus and CNS isolates, respectively. Resistance to penicillin was attributed to the presence of blaZ, erythromycin resistance to ermC (alone or combined with ermB) and tetracycline resistance to tetK (alone or combined with tetM). Notably, one S. equorum isolate and one S. saprophyticus isolate were both methicillin-resistant and mecA positive. Additionally, all S. aureus isolates carried the adhesin genes fnbpA, clfA, clfB, and sdrC, and most of them contained cna and sdrE. Conversely, only a few of the CNS isolates carried clfA, cna, and fnbA. Regarding toxin genes, all S. aureus isolates harbored hlb, and most of them were hlg positive. The lukE-lukD, lukM, sec, sed, sei, sen, seo, tst, seg, seh, and sej were also detected with low frequencies. However, no toxin genes were observed in CNS isolates. This study reveals high species diversity of staphylococci from clinical bovine mastitis in Ningxia Hui Autonomous Region of China. The findings for the genetic determinants of antimicrobial resistance and virulence factor provide valuable information for control and prevention of staphylococcal bovine mastitis.

Comparative Genomic Analyses of Lactococcus garvieae Isolated from Bovine Mastitis in China.

Lactococcus garvieae is an emerging zoonotic pathogen, but there are few reports regarding bovine mastitis. The prevalence of L. garvieae represents an increasing disease threat and global public health risk. Thirty-nine L. garvieae isolates were obtained from 2,899 bovine clinical mastitis milk samples in 6 provinces of China from 2017 to 2021. Five clonal complexes were determined from 32 multilocus sequence types (MLSTs) of L. garvieae: sequence type 46 (ST46) was the predominant sequence type, and 13 novel MLSTs were identified. All isolates were resistant to chloramphenicol and clindamycin, but susceptible to penicillin, ampicillin, amoxicillin-clavulanic acid, imipenem, ceftiofur, enrofloxacin, and marbofloxacin. Based on genomic analyses, L. garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase. Most isolates had lsaD and mdtA antimicrobial resistance (AMR) genes. Based on COG (Clusters of Orthologous Genes database) results, the functions of defense, transcription and replication, and recombination and repair were enhanced in unique genes, whereas functions of translation, ribosomal structure, and biogenesis were enhanced in core genes. The KEGG functional categories enriched in unique genes included human disease and membrane transport, whereas COG functional categories enriched in core genes included energy metabolism, nucleotide metabolism, and translation. No gene was significantly associated with host specificity. In addition, analysis of core genome single nucleotide polymorphisms (SNPs) implied potential host adaptation of some isolates in several sequence types. In conclusion, this study characterized L. garvieae isolated from mastitis and detected potential adaptations of L. garvieae to various hosts. IMPORTANCE This study provides important genomic insights into a bovine mastitis pathogen, Lactococcus garvieae. Comprehensive genomic analyses of L. garvieae from dairy farms have not been reported. This study is a detailed and comprehensive report of novel features of isolates of L. garvieae, an important but poorly characterized bacterium, recovered in the past 5 years in 6 Chinese provinces. We documented diverse genetic features, including predominant sequence type ST46 and 13 novel MLSTs. Lactococcus garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase and resistance to chloramphenicol and clindamycin. Most isolates had lsaD and mdtA antimicrobial resistance genes. However, no gene was significantly associated with host specificity. This is the first report that characterized L. garvieae isolates from bovine mastitis and revealed potential host adaptations of L. garvieae to various hosts.

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.

Comparative Genomic Analysis of Streptococcus dysgalactiae subspecies dysgalactiae Isolated From Bovine Mastitis in China.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is one of the most prevalent pathogens causing bovine mastitis worldwide. However, there is a lack of comprehensive information regarding genetic diversity, complete profiles of virulence factors (VFs), and antimicrobial resistance (AMR) genes for SDSD associated with bovine mastitis in China. In this study, a total of 674 milk samples, including samples from 509 clinical and 165 subclinical mastitis cases, were collected from 17 herds in 7 provinces in China from November 2016 to June 2019. All SDSD isolates were included in phylogenetic analysis based on 16S rRNA and multi-locus sequence typing (MLST). In addition, whole genome sequencing was performed on 12 representative SDSD isolates to screen for VFs and AMR genes and to define pan-, core and accessory genomes. The prevalence of SDSD from mastitis milk samples was 7.57% (51/674). According to phylogenetic analysis based on 16S rRNA, 51 SDSD isolates were divided into 4 clusters, whereas based on MLST, 51 SDSD isolates were identified as 11 sequence types, including 6 registered STs and 5 novel STs (ST521, ST523, ST526, ST527, ST529) that belonged to 2 distinct clonal complexes (CCs) and 4 singletons. Based on WGS information, 108 VFs genes in 12 isolates were determined in 11 categories. In addition, 23 AMR genes were identified in 11 categories. Pan-, core and accessory genomes were composed of 2,663, 1,633 and 699 genes, respectively. These results provided a comprehensive profiles of SDSD virulence and resistance genes as well as phylogenetic relationships among mastitis associated SDSD in North China.

Genetic Polymorphisms of Staphylococcus aureus Isolated from Bovine Mastitis in China

Genetic Polymorphisms of Staphylococcus aureus Isolated from Bovine Mastitis in China

In vitro immune responses of bovine mammary epithelial cells induced by Escherichia coli, with multidrug resistant extended-spectrum β-lactamase, isolated from mastitic milk

Bovine mastitis is an inflammatory condition of mammary glands causing huge economic losses for dairy industries. Infection with extended-spectrum β-lactamase (ESBL)-producing sequence types (ST) 410-Escherichia coli (ESBL-ST410 E. coli) is considered a leading cause of bovine mastitis in China. However, pathogenic effects of these strains in an in vitro model, e.g. bovine mammary epithelial cells (bMECs), are unknown. Therefore, our objectives were to explore pathogenesis (adhesion and invasion, inflammation, oxidative stress and apoptosis) of ESBL-E. coli (highly prevalent in bovine mastitis) in bMECs. Non-pathogenic E. coli DH5α and a prototypical E. coli P4 were included as negative and positive controls, respectively. The bMECs were infected with our isolated ST410 strains, plus DH5α and P4, with assessment of the following end points: adhesive and invasive capabilities; lactate dehydrogenase (LDH) activities; inflammatory responses, including concentrations of interleukin-1β (IL-1β), IL-6, IL-10 and tumor necrosis factor-α; oxidative stress including intracellular reactive oxygen species production, malondialdehyde concentrations, activities of glutathione peroxidase and superoxide dismutase; and apoptosis. All ST410 strains had greater adhesive and invasive capabilities and increased LDH release, with varying degrees of inflammatory responses, oxidative stress and apoptosis compared to blank and DH5α groups, similar to P4-infected bMECs. In particular, ST410(4) was more likely than the other 3 isolates to adhere to and invade bMECs and increase LDH activities, cytokine release, oxidative stress and apoptosis. Thus, ST410 isolates had pathogenic manifestations of adhesive and invasive capabilities; furthermore, they induced inflammation, oxidative stress and apoptosis in bMECs. Finally, ST410(4) was the most pathogenic strain.

Mycoplasma bovis-generated reactive oxygen species and induced apoptosis in bovine mammary epithelial cell cultures

Mycoplasma bovis is an important cause of bovinemastitis in China and worldwide. We hypothesized that M. bovis damages bovine mammary epithelial cells (bMEC), with the degree of damage varying among field isolates. Our objective was to evaluate 2 novel sequence type (ST) field strains of M.bovis (ST172 and ST173) for their ability to induce oxidative stress, cytotoxicity, pathomorphological changes, and apoptosis in bMEC, as a model for pathogenesis of M. bovis-induced bovine mastitis. Cytotoxicity (as indicated by release of lactate dehydrogenase, LDH) from bMEC depended on multiplicity of infection (MOI), with a high MOI (1:1,000) being required to induce cytotoxicity. Morphological changes in bMEC, including shrinkage, loss of cell integrity, and heavy staining (hematoxylin and eosin) of cytoplasm were apparent 24 h after infection with ST172 or ST173 M.bovis, with more severe changes being induced by the latter strain. Adhesion and invasion assays both had curvilinear patterns, peaking 12 h after infection with MOI of 1:1,000. Both production of reactive oxygen species (ROS) and proportion of apoptotic cells increased with time after infection. Increased Bax/Bcl-2 ratios and activation of caspase-3 implied involvement of mitochondria-dependent pathways ofapoptosis. Furthermore, intracellular ROS generation, apoptosis, and cleaved caspase-3 were mitigated by N-acetyl-l-cysteine, a ROS scavenger. Both interleukin (IL)-1β and IL-6 were significantly upregulated by ST172 and ST173 M. bovis, with little change in expression of tumor necrosis factor-α. One ST173 M. bovis isolate had the greatest cytotoxicity of all of our field isolates, with the highest LDH release, adhesion, invasion, ROS production, and apoptosis. In conclusion, our hypothesis was supported: M. bovis damaged bMEC by generating ROS and initiating a mitochondria-dependent pathway ofapoptosis, with the degree of damage varying among field isolates. This study provided new knowledge regarding pathogenesis of M. bovis-induced bovine mastitis.

Short communication: Detection and molecular characterization of methicillin-resistant Staphylococcus aureus isolated from subclinical bovine mastitis cases in China

This study investigated the antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) isolated from cases of subclinical bovine mastitis in China, as well as resistance mechanisms and virulence genes encoding adhesins and toxins. We determined antimicrobial susceptibility using the disk diffusion method, and analyzed resistance, adhesin, and toxin genes using PCR. We confirmed MRSA in 73 of 498 (14.7%) Staph. aureus isolates recovered from subclinical mastitic milk samples. All isolates were positive for mecA. The MRSA isolates showed high resistance to penicillin (100.0%), gentamicin (100.0%), and tetracycline (98.6%). All MRSA isolates harbored resistance genes blaZ (penicillin), aacA/aphD (gentamicin), and tetM (alone or in combination with tetK, tetracycline). Moreover, all isolates carried the adhesin genes fnbpA, clfA, clfB, cna, sdrE, and map/eap, and most carried sdrC (98.6%), sdrD (95.9%), bbp (94.5%), and ebpS (80.8%). The toxin genes seh, hla, and hld were present in all isolates, and most isolates carried sea (71.2%), seg (84.9%), sei (82.2%), lukE-lukD (97.3%), and hlg (72.6%). These findings of high-level resistance to antimicrobials commonly used in dairy cattle should lead to calls for antibiogram analysis before antimicrobial therapy. The high frequency of adhesin and toxin genes in MRSA indicates their potential virulence in bovine mastitis in China.

Characteristics of quinolone-resistant Escherichia coli isolated from bovine mastitis in China

Escherichia coli is the leading causative agent of bovine mastitis worldwide. Quinolone-resistant E. coli is becoming a potential threat to veterinary and public health. The aim of this study was to investigate the characteristics of quinolone-resistant E. coli isolated from bovine mastitis cases in China. Antimicrobial susceptibility of the isolates against 15 antimicrobial agents was determined by disc diffusion method. Phylogenetic grouping was detected by PCR. Extended-spectrum β-lactamase-producing isolates were determined by double-disc synergy test. In addition, the plasmid-mediated quinolone resistance (PMQR) and β-lactamase-encoding genes, as well as mutations of quinolone resistance-determining regions in GyrA, GyrB, ParC, and ParE, were measured by PCR and DNA sequencing. Overall, 75 (22.9%) out of 328 E. coli isolates were confirmed as ciprofloxacin-resistant from 2,954 mastitic milk samples. Phylogenetic group analysis showed that the majority of these strains belonged to phylogenetic group A (57.3%) and group B1 (24.0%). All the resistant isolates were identified as multidrug resistant, showing high resistance to cephalosporins and non-β-lactams. Forty-nine (65.3%) of the quinolone-resistant isolates were positive for PMQR genes; aac-(6')-Ib-cr was the most common PMQR determinant detected in 33 (44.0%) isolates. Eighteen (24.0%), 4 (5.3%), 3 (4.0%), and 1 (1.3%) of the quinolone-resistant isolates were harboring oqxA/B, qepA4, qnrS, and qnrB2, respectively. Additionally, 55 (73.3%) of the quinolone-resistant E. coli isolates were found to be extended-spectrum β-lactamase producers. The preponderant β-lactamase-encoding gene, blaTEM, was detected in 44 (58.7%) isolates; blaCTX-M, blaCMY, and blaSHV were found in 35 (46.7%), 22 (29.3%), and 2 (2.7%) isolates, respectively. Moreover, the most frequently identified substitutions were S83L/D87N or S83L in GyrA, detected in all of the quinolone-resistant isolates. Meanwhile, 74 (98.7%), 33 (44.0%), and 6 (8.0%) of the isolates were carrying substitutions S80I in ParC, S458A in ParE, and S492N in GyrB, respectively. All 58 (77.3%) isolates with a high level of ciprofloxacin resistance (>32 µg/mL) carried single or double mutations in GyrA combined with single mutation in ParC. To the best of our knowledge, this is the first report on the high occurrence of PMQR determinants and quinolone-determining resistant regions mutations in quinolone-resistant E. coli isolated from bovine mastitis in China.

Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China

BackgroundStaphylococcus aureus, a common cause of bovine mastitis, is known for its ability to acquire to antimicrobial resistance and to secrete numerous virulence factors that can exacerbate inflammation. In addition, alpha-hemolysin has an important role in S. aureus infections, diversity of the hla gene (that produces alpha-hmolysin) in S. aureus isolated from bovine mastitis has not been well characterized. The objective was, therefore, to determine diversity of virulence genes, hla gene sequences, and clonal profiles of S. aureus from bovine mastitis in Chinese dairy herds, and to evaluate inter-relationships.ResultsThe antimicrobials resistance varies from as low as 1.9% (2/103) for CTX to as high as 76.7% (79/103) for penicilin in the 103 isolates and 46 (44.7%) S. aureus were determined as multi-resistant isolates with diverse resistance patterns. Thirty-eight virulence gene patterns (with variable frequencies) were identified in the 103 isolates and correlated with MLST types, indicating a great diversity. Although the hla gene also had great diversity (14 genotypes), Hla peptides were relatively more conserved. With 7 clonal complexes identified from 24 spa types and 7 MLST types. Regarding the letter, ST 97 was the dominant type in S. aureus from bovine mastitis in China. Furthermore, based on phylogenetic analysis, there was a distinct evolutionary relationship between the hla gene and MLST.ConclusionMulti-resistant S. aureus occurred in bovine mastitis with diverse resistance patterns. The diversity of virulence gene profiles, especially the hla gene and, their relationship with molecular types were reported for the first time in S. aureus from bovine mastitis, which will be useful for future studies on immunogenicity and vaccine development. In addition, based on the distinct evolutionary relationship between the hla gene and MLST types, we inferred that the hla gene has potential role for molecular typing of S. aureus.

Short communication: Molecular characteristics, antimicrobial susceptibility, and pathogenicity of clinical Nocardia cyriacigeorgica isolates from an outbreak of bovine mastitis

The occurrence of nocardial mastitis, mostly in the context of outbreaks, has been reported in many countries. However, there is a paucity of reports regarding detailed characterization of Nocardia cyriacigeorgica from bovine mastitis. Thus, herein we report characteristics, antimicrobial susceptibility patterns, molecular identification, and pathogenicity of N. cyriacigeorgica isolated from an outbreak of clinical mastitis in a dairy herd in northern China. A total of 182 (80.2%) lactating cows had clinical mastitis with severe inflammation and firmness of the udder, reduced milk production, and anorexia, with no apparent clinical response to common antibiotics. Out of 22 mastitic milk samples submitted to our laboratory, 12 N. cyriacigeorgica were isolated and characterized using standard microbiological analysis, 16S rRNA gene sequencing, random amplified polymorphic DNA PCR analysis, biochemical assays, and antibiotic susceptibility testing. Additionally, in vivo experiments were done to determine pathogenicity of these clinical mastitis isolates. All isolates were resistant to ampicillin, amoxicillin-clavulanic acid, ciprofloxacin, minocycline, rifampicin, and aminoglycosides (type VI pattern). Additionally, intramammary inoculation of mice with N. cyriacigeorgica caused chronic inflammatory changes, including hyperemia, edema, and infiltration of lymphocytes and neutrophils, as well as hyperplasia of lymph nodules in mammary glands. Therefore, we concluded that N. cyriacigeorgica was involved in the current outbreak of mastitis. To our best knowledge, this is the first report to characterize N. cyriacigeorgica isolated from cases of bovine mastitis in China.

Population structure and antimicrobial profile of Staphylococcus aureus strains associated with bovine mastitis in China

Staphylococcus aureus is a significant bacterial pathogen associated with bovine mastitis. The aim of the present study was to investigate and characterize of S. aureus strains isolated from the milk of cows suffering from mastitis in the mid-east of China. Among the 200 milk samples analyzed, 58 were positive for S. aureus, of these isolates, 11 isolates were methicillin-resistant Staphylococcus aureus (MRSA). All of the 58 S. aureus strains were classified in agr group I, while seven different sequence type (ST) patterns were identified and among them the most common was ST630 followed by ST188. All of the S. aureus isolates belonging to ST630 were resistant to more than four antimicrobials, and 22.2% of isolates belonging to ST188 were resistant to eight antimicrobials. Interestingly, while strong biofilm producers demonstrated higher resistance to multiple antimicrobials, they exhibited lower intracellular survival rates. The results of this study illustrated the distribution, antimicrobial susceptibility profiles, genotype, and the ability of biofilm production and mammary epithelial cells invasion of these S. aureus isolates. This study can provide the basis for the development of a disease prevention program in dairy farms to reduce the potential risk in both animal and human health.

Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China.

Trueperella (T.) pyogenes is an opportunistic pathogen that causes suppurative diseases in domestic animals. In this work, the properties, pathogenesis and phenotypic diversity of T. pyogenes isolates from bovine mastitis were studied. Both pyolysin (plo) and collagen-binding protein (cbp) virulence factor genes were detected by PCR in all T. pyogenes isolates (n = 50). Using the tissue culture plate method, 90% of T. pyogenes isolates were able to form biofilms. The minimum inhibitory concentrations (MICs) of 13 antimicrobials against T. pyogenes isolates were determined. High susceptibility was observed to rifampin (96%), ampicillin (94%), ciprofloxacin (94%), and penicillin (92%), while low susceptibility was found to trimethoprim-sulphamethoxazole (10%) and bacitracin (2%). The intracellular assay revealed that T. pyogenes isolates had different cytopathogenic effects on cells. The high percentage (28.6%) of T. pyogenes isolates suggests that this bacterium is an important contributor to mastitis. Moreover, the high occurrence of multidrug resistance, biofilm production, intracellular survival, and the temporal dynamics of T. pyogenes interactions are key factors for a better understanding of how immunity acts on infections with these bacteria and how they evade immune surveillance, thus highlighting the need for the prudent use of antimicrobial agents in veterinary medicine.

Characterization of Prototheca zopfii Genotypes Isolated from Cases of Bovine Mastitis and Cow Barns in China.

Protothecal mastitis, caused mostly by Prototheca zopfii (P. zopfii), is increasing in dairy herds and is being reported globally. The present study was aimed at studying the epidemiology of mastitis and at molecular characterization of P. zopfii isolates from dairy herds and their surroundings in three provinces of China using microbiological, biochemical and molecular methods, and antibiotic susceptibility tests. Samples from milk (n = 620) of mastitic cows and their barns sources (n = 410) including feces, feed, bedding materials and drinking water were analyzed. Among other pathogens recovered from mastitic milk, 84 (13.5%) of the isolates were identified as P. zopfii. All of the P. zopfii isolates recovered from milk were recognized as genotype 2, whereas 58 (73.4%) and 21 (26.6%) isolates from environmental sources were found to be P. zopfii genotypes 1 and 2, respectively. The isolates were susceptible to some antibiotics and antifungal agents, including amikacin (78.1%), streptomycin (58.5%), gentamicin (17.8%), amphotericin B (68.6%) and nystatin (64.4%). Additionally, the two genotypes displayed versatile patterns of susceptibility to different antimicrobials agents. Phylogeny of the genotypes on the basis of 18S SSU rDNA and 28S SSU rDNA was also investigated. The isolates of the two genotypes separated into different clades, and no interrelationship was observed among these as shown by phylogenetic analysis. The genotype 1 isolates from cow barn sources were non-pathogenic and may not present any risk of mastitis. We conclude that P. zopfii genotype 2 might play an important role in bovine mastitis in China.

Evaluation of analgesic and anti-inflammatory activities of compound herbs Puxing Yinyang San

Background: Bovine mastitis is one of the most relevant and problematic diseases to treat and control in practice. Puxing Yinyang San (PYS) is a compound of herbs to treat bovine mastitis in China. This study was performed to evaluate the analgesic and anti-inflammatory activities of PYS in mice and rats.Materials and Methods: The analgesic and anti-inflammatory activities of PYS were determined using acetic acid-induced writhing response, hot plate test, xylene-induced ear swelling test, carrageenan-induced paw edema test, and acetic acid-induced capillary permeability and leukocyte infiltration test with oral doses of 155, 310 and 620 mg/kg·bw in mice or rats.Results: The acetic acid-induced writhing response was dose-dependently inhibited by oral administration of PYS and the latency time to thermal stimuli was increased in the hot plate test, especially 90 minutes after treatment. In the xylene-induced ear swelling, PYS significantly decreased swelling degree in a dose-dependent manner. Additionally, PYS significantly suppressed the peritoneal capillary permeability and leukocyte infiltration in mice induced by intraperitoneal injection of acetic acid. PYS also significantly reduced the carrageenan-induced rat paw edema at 2, 3, and 4 h after the carrageenan injection. The results suggested that PYS possessed significant analgesic and anti-inflammatory activities.Conclusion: This study was the first to demonstrate that oral administration of PYS might play an important role in the process of analgesia and anti-inflammation, supporting its treatment for mastitis. Future investigations will focus on the broader involvement of the ingredients and mechanisms responsible for pharmacological activities of PYS.Keywords: Puxing Yinyang San, analgesic activity, anti-inflammatory activity, herb.

Prevalence of 16S rRNA Methylase Gene rmtB Among Escherichia coli Isolated from Bovine Mastitis in Ningxia, China.

The aim of this study is to understand the prevalence and molecular characterization of 16S rRNA methylase gene, rmtB, among Escherichia coli strains isolated from bovine mastitis in China. A total of 245 E. coli isolates were collected from bovine mastitis in China between 2013 and 2014 and were screened for 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA) by polymerase chain reaction. About 5.3% (13/245) of the isolates carried the rmtB gene; the isolates were highly resistant to amikacin. Thirteen rmtB-positive strains were analyzed for the presence of extended-spectrum β-lactamase genes (bla(TEM), bla(CTX-M), bla(OXA), and bla(SHV)). All the isolates harbored both bla(TEM-1) and bla(CTX-M-15) genes and two of the isolates were also positive for bla(OXA-1). Pulsed-field gel electrophoresis (PFGE) analysis indicated that the nine rmtB-positive strains belonging to ST10 from one farm showed the similar PFGE pattern, indicating a clonal expansion in this farm. S1-PFGE and Southern blotting showed that 12 isolates harbored the rmtB gene in plasmids of two different sizes (≈45 kb [n=10] and ≈48 kb [n=2]), while only 1 strain harbored the rmtB gene in the chromosome. These plasmids were transferable by conjugation studies, and two isolates from two respective farms carried the same size of plasmid, suggesting that the horizontal transmission of plasmids also contributed to the spread of rmtB gene. This is the first report of prevalence of the 16S rRNA methylase gene rmtB among E. coli isolated from bovine mastitis in China, and rmtB-carrying E. coli may pose a threat to the treatment of bovine mastitis.

EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITIES OF COMPOUND HERBS PUXING YINYANG SAN

Background: Bovine mastitis is one of the most relevant and problematic diseases to treat and control in practice. Puxing Yinyang San (PYS) is
 a compound of herbs to treat bovine mastitis in China. This study was performed to evaluate the analgesic and anti-inflammatory activities of
 PYS in mice and rats.
 Materials and Methods: The analgesic and anti-inflammatory activities of PYS were determined using acetic acid-induced writhing response,
 hot plate test, xylene-induced ear swelling test, carrageenan-induced paw edema test, and acetic acid-induced capillary permeability and
 leukocyte infiltration test with oral doses of 155, 310 and 620 mg/kg·bw in mice or rats.
 Results: The acetic acid-induced writhing response was dose-dependently inhibited by oral administration of PYS and the latency time to
 thermal stimuli was increased in the hot plate test, especially 90 minutes after treatment. In the xylene-induced ear swelling, PYS significantly
 decreased swelling degree in a dose-dependent manner. Additionally, PYS significantly suppressed the peritoneal capillary permeability and
 leukocyte infiltration in mice induced by intraperitoneal injection of acetic acid. PYS also significantly reduced the carrageenan-induced rat paw
 edema at 2, 3, and 4 h after the carrageenan injection. The results suggested that PYS possessed significant analgesic and anti-inflammatory
 activities.
 Conclusion: This study was the first to demonstrate that oral administration of PYS might play an important role in the process of analgesia and
 anti-inflammation, supporting its treatment for mastitis. Future investigations will focus on the broader involvement of the ingredients and
 mechanisms responsible for pharmacological activities of PYS.