Influence of 17beta-estradiol, progesterone, and dexamethasone on diapedesis and viability of bovine blood polymorphonuclear leukocytes

The aim of the current study was to investigate whether polymorphonuclear leukocyte (PMN) diapedesis and viability are influenced by steroid hormones. Using an in vitro model with different types of cell layers (bovine mammary epithelial cells and fibroblasts), we investigate whether steroid hormone treatments (17beta-estradiol, progesterone, and dexamethasone) have an influence on the diapedesis capacity and viability of PMN. In addition, we studied apoptosis of PMN in the in vitro model and evaluated the influence of different types of cell layers and steroid hormone treatments on this process. A significant decrease in the number of viable PMN in the lower compartment of the in vitro model (i.e., number of migrated PMN x viability after migration) was found after 17beta-estradiol treatment, whereas no influence was detected after progesterone or dexamethasone treatment. The effect of 17beta-estradiol was not due to a lower viability before migration as none of the treatments caused a significant effect on the viability before diapedesis. This treatment effect was not influenced by endogenous 17beta-estradiol or progesterone levels before isolation because there was no correlation between these plasma levels and PMN diapedesis capacity or viability. Furthermore, migration through epithelial cells caused a significant decrease in viability of PMN due to increased apoptosis but not necrosis.     

Association of bovine leukocyte antigen (BoLA) DRB3.2 with immune response, mastitis, and production and type traits in Canadian Holsteins

Data collected from 328 Canadian Holsteins in a research herd at the University of Guelph were used to study associations among expression of bovine leukocyte antigen (BoLA) DRB3.2 alleles, immune response, mastitis resistance via somatic cell counts (SCC), and clinical mastitis, as well as to extend these results to production and type traits. Accordingly, groups of cows were evaluated in vivo for both the antibody-mediated immune response (AMIR) and the cell-mediated immune response (CMIR), which generally predominate in responses to extracellular and intracellular pathogens, respectively. Of note was that associations between BoLA DRB3.2 alleles and immune responses tended to be in the opposite sign for the 2 AMIR and CMIR traits examined. For example, alleles DRB3.2*3 and *24 were associated with higher AMIR but lower CMIR, whereas allele *22 was associated with lower AMIR but higher CMIR. This finding is in agreement with the hypothesis that both traits are genetically independent and represent opposing type 1 and type 2 immune responses. Additionally, BoLA DRB3.2*3 and *11 were associated with lower SCC, whereas alleles *22 and *23 were associated with higher SCC. Finally, allele DRB3.2*3 was also associated with less clinical mastitis, whereas allele *8 was associated with higher mastitis risk. Allele *3 was of particular relevance because it was associated with increased antibodies, as well as reduced mastitis and SCC. This could be due to an indirect relationship between the ability to produce a high antibody response and enhanced defense against intrammamary infections caused by extracellular pathogens. Consequently, the BoLA DRB3.2*3 allele should be investigated further as a candidate for resistance to some types of intramammary infections, the important caveat being its association with lower CMIR, particularly with one of the test antigens used to evaluate delayed-type hypersensitivity. The results of associations between BoLA DRB3.2 and production traits were, in some cases, antagonistic in that BoLA DRB3.2 alleles *11 and *23, which are associated with increased production traits, were associated with lower and higher SCC, respectively. Collectively, these findings advocate the use of alleles *3, *23, and *22 as reference points for more detailed mechanistic studies. This does not imply that genetic selection for mastitis resistance should be based on BoLA alleles, but that information on a variety of genes may aid in identification and selection for improved health.     

Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification

The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) label-free semiquantitative approach. Liquid chromatography-MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60 h following infection resulted in the identification of the high- to medium-abundance proteins α_S1-, α_S2- β-, and κ-caseins and the whey proteins serum albumin, β-lactoglobulin, and α-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC-MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC-MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists.     

Methionine and choline supply during the peripartal period alter polymorphonuclear leukocyte immune response and immunometabolic gene expression in Holstein cows

Polymorphonuclear leukocytes (PMNL) are the first responders upon pathogen invasion and hence play an important role in inflammatory and immune responses. Rumen-protected methionine (MET) and choline (CHOL) during the peripartal period affect the immune response and inflammatory status in dairy cows to different extents. We aimed to examine the effect of MET and CHOL supply on expression of genes regulating key PMNL functions and associations with whole-blood immune challenge. Thirty multiparous Holstein cows from a larger cohort randomly assigned from ?21 to 30 d relative to parturition to a basal control (CON) diet, CON plus MET at a rate of 0.08% of dry matter, or CON plus CHOL at 60 g/d were used. Blood was sampled at ?10, 7, and 30 d relative to parturition for inflammatory biomarker analyses and PMNL isolation. Neutrophil and monocyte phagocytosis and oxidative burst in vitro were assessed in whole blood at 1, 7, and 28 d. Although neutrophil and monocyte phagocytosis did not differ, oxidative burst in neutrophils and monocytes was greater in MET-supplemented cows relative to CON cows. Compared with CON, PMNL adhesion and migration-related genes (ITGAM, ITGB2, ITGA4) were downregulated in response to MET and CHOL. Expression of CADM1 and SELL was also lower in MET-supplemented cows compared with CON cows but not in CHOL cows. In contrast, compared with CON cows, the expression of ICAM1 was lower in CHOL but not MET cows. Similar to adhesion and migration-related genes, cows receiving MET- or CHOL-supplemented diets had lower expression of inflammation-related genes (IL1β, IL10RA, NFKB1, STAT3, TLR2). However, expression of IRAK1 and TLR4 was lower in MET- but not CHOL-supplemented cows. Plasma taurine concentration was greater in MET cows compared with CHOL and CON cows, suggesting a better redox status in plasma. In agreement with plasma taurine, oxidative stress-related genes (CBS, CTH, GPX1, GSS, SOD2) in PMNL were lower in response to MET and to CHOL supply. Overall, immunometabolic gene expression profile and blood biomarker analyses suggest an overall better redox status in PMNL during the transition period in response to MET and CHOL supply. These adaptations in PMNL might be beneficial for mounting a better bactericidal response upon challenge.     

Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea

Staphylococcus aureus belongs to the group of major contagious mastitis pathogens, whereas the coagulase-negative staphylococci (CNS) are also capable of causing opportunistic bovine mastitis. Many of these strains are resistant to penicillin or ampicillin because of the long-term use of β-lactam antibiotics in agricultural and healthcare settings. Based on the simple and highly specific coagulase genotyping by PCR-RFLP used for discriminating among Staph. aureus strains, the relationship between phenotypic antibiogram and the polymorphism of coagulase gene was determined in this study. The staphylococci strains (835 Staph. aureus and 763 CNS) were isolated from 3,047 bovine mastitic milk samples from 153 dairy farms in 8 provinces from 1997 to 2004 in the Republic of Korea. Twenty-one (2.5%) Staph. aureus and 19 (2.4%) CNS strains were resistant to methicillin [oxacillin minimum inhibitory concentration (MIC) ≥4 μg/mL]. The mecA gene was also found in 13 methicillin-resistant Staph. aureus (MRSA) and 12 methicillin-resistant CNS (MRCNS) isolates with a significantly higher detection rate of the mecA gene in MRSA with high MIC (≥16 μg/mL) compared with those with MIC ≤ 8 μg/mL. Methicillin-resistant Staph. aureus and MRCNS were also more resistant to other antibiotics (ampicillin, cephalothin, kanamycin, and gentamicin) than methicillin-susceptible staphylococci. Among 10 different coa PCR-RFLP patterns (A to J) in 706 Staph. aureus strains, the main types were A (26.9%), B (17.0%), G (10.5%), and H (15.4%), with the frequent observation of the A and H types (6 and 10 isolates) in MRSA. This study indicates that major epidemic Staph. aureus clones may be spread between different dairy farms, and the profile of coa genotype can be applied for epidemiological investigations and control of bovine mastitis, particularly one caused by MRSA with specific prevalent coa types.     

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap−), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.     

L-selectin and chemotaxis throughout bone marrow granulocyte maturation in the bovine

Polymorphonuclear neutrophilic leukocytes (PMNL) play a pivotal role during inflammation. Bone marrow (BM) reserves are depleted as cells are released into circulation for recruitment to infection sites. Expression of L-selectin on the cell membrane allows neutrophils to roll along the activated endothelium. Whereas mechanisms leading to recruitment to infection sites are well established, expression of BM adhesion molecules in cows is limited. In this study, we assessed L-selectin expression and chemotactic response to zymosan-activated serum (ZAS) in bovine BM cells and in circulating neutrophils. Isolated blood PMNL and BM cells were used from 9 dairy cows, for quantifying L-selectin expression using flow cytometry, and from 12 dairy cows for chemotaxis studies. All granulocytic maturation stages expressed L-selectin. The percentage of cells fluorescing increased significantly in BM band and mature granulocytes and reached maximal expression on circulating neutrophils. Bone marrow band and segmented cells showed the highest L-selectin density. Chemotaxis through micropore filters in response to zymosan-activated fetal bovine serum was first observed in the myelocytic and metamyelocytic stages, and it increased with maturation and release into the blood stream. From these results, we conclude that L-selectin expression varies among stages of granulocytic maturation within the BM and differs from circulating PMNL. Further, BM cells are capable of migration starting at the metamyelocytic stage, and compared with BM cells, circulating neutrophils are more chemotactively active.     

Short communication: Antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China

This study aimed to investigate the antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China. Enterococcus faecalis isolates were identified by 16S rRNA amplification and sequencing. Antimicrobial susceptibility was determined by the disc diffusion method. Antimicrobial resistance and virulence genes were tested by PCR. Overall, E. faecalis was recovered from 81 of 1,787 (4.5%) mastitic milk samples. The isolates showed high resistance against tetracycline (87.7%) and erythromycin (79.0%). The most prevalent resistance genes found in the E. faecalis were tetK (96.3%), tetL (79.0%), and tetM (87.7%) for tetracycline and ermC (97.5%) for erythromycin. Moreover, gelE (70.4%), esp (85.2%), efaA (91.4%) were the most common virulence genes. This is the first report to characterize E. faecalis recovered from subclinical bovine mastitis cases in China.     

Relationships among superantigen toxin gene profiles,genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.     

Short communication: Mycolicibacterium smegmatis,basonym Mycobacterium smegmatis,causing pyogranulomatous mastitis and its cross-reactivity in bovine (para)tuberculosis testing

Different mycobacterial species are encountered in bovine medicine. The fastidiously growing mycobacteria (Mycobacterium bovis as the cause of bovine tuberculosis, and Mycobacterium avium ssp. paratuberculosis, MAP, as the cause of paratuberculosis) are well known and targeted in eradication/control or monitoring programs in different countries, whereas the rapidly growing species is only rarely identified from bovine disease. The latter have occasionally been reported as the cause of bovine clinical mastitis, but recent reports are scarce. In this study, Mycolicibacterium smegmatis (basonym Mycobacterium smegmatis) was identified as cause of granulomatous, relapsing clinical mastitis in 2 cows from one Belgian dairy herd. Milk, blood, and fecal samples were collected, as well as tissue samples after the cows were culled. Serological analysis conducted on milk and serum samples resulted in positive reactions for MAP, but negative for Mycobacterium bovis. Production of IFN-γ showed sensitization with mycobacteria or similar organisms, other than M. bovis, in one cow. Detection of MAP by bacteriological culture and IS900-based quantitative PCR on milk and feces remained negative. In conclusion, this paper describes M. smegmatis as a cause of bovine clinical mastitis in Belgium and suggests cross-reactivity of the intramammary M. smegmatis infection with routinely used serological tests for MAP.     

Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility

Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of “commensal mammary microbiota,” the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.     

Antibiogram and coagulase diversity in staphylococcal enterotoxin-producing Staphylococcus aureus from bovine mastitis

We investigated antibiogram and coagulase gene diversity in staphylococcal enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk samples of cows infected with mastitis from 140 dairy farms in Korea between 1997 and 2004. Of the 696 Staph. aureus isolates collected in this study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant. The percentage of StE-producing Staph. aureus (SES) isolates resistant to methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater than that of non-SES. Ten coagulase genotype patterns were observed, including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and VIII (17.8%). More than 4 Staph. aureus types were isolated from each of 82 dairy farms in different geographic locations, and only 1 coagulase genotype pattern was observed in 39 of the herds (47.6%). There was no significant correlation between coagulase genotypes harbored by Staph. aureus and their specific StE type. The percentage of isolates producing major StE types (A, B, AC, and ABCD) and being resistant to cephalothin and methicillin was greater among the Staph. aureus isolates with the 4 predominant coagulase genotypes (I, II, VII, and VIII) than among the isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X). Based on coagulase gene polymorphisms, our data indicate that a broad distribution of identical or closely related enterotoxin-producing Staph. aureus strains seem to contribute to bovine mastitis in the Republic of Korea.     

The phylogenetic group,antimicrobial susceptibility,and virulence genes of Escherichia coli from clinical bovine mastitis

Bovine mastitis is still a central problem on dairy farms despite control programs, and Escherichia coli is a crucial pathogen during the development of bovine mastitis. The virulence genes, antimicrobial susceptibility, and mortality of mice infected with different E. coli isolates from bovine mastitis were determined in this study. According to the presence of the specific genes chuA, yjaA, and TspE4.C2, these isolates mainly belonged to 2 different groups: group A (47/79) and group B1 (22/79). The ompC gene was detected in all the isolates, followed by fimH (89.9%), ECs3703 (88.6%), and ompF (73.4%), whereas most of the virulence genes were not detected in these isolates. The results of the antimicrobial susceptibility tests indicated that the isolates were susceptible to the fluoroquinolones and aminoglycosides. An inverse relationship was shown between the expression level of ompF and antimicrobial resistance; additionally, the isolates that were nonsusceptible to at least 4 classes of antimicrobial agents showed a lower mortality to mice in comparison with the susceptible isolates. This study indicated that antibiotic resistance had emerged in E. coli from bovine mastitis in this area, and appropriate measures should be taken to avoid potential threats to humans and other animals.     

Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal

Methicillin-resistant staphylococci (MRS) have already been reported as mastitis agents. Such bacterial species are a public health concern, and the characterization of their antimicrobial resistance and virulence profile is important to better control their dissemination. The present work evaluated the distribution of methicillin-resistance among 204 staphylococci from clinical (n = 50) and subclinical (n = 154) bovine mastitis. The presence ofthe mecA gene was determined by PCR. Phenotypic expression of coagulase, DNase, lipase, gelatinase, hemolytic enzymes, and biofilm production was evaluated. The presence of biofilm-related genes, icaA, icaD, and bap, was also determined. Antimicrobial resistance patterns for aminoglycosides, lincosamides, macrolides, fluoroquinolones, sulphonamides, tetracyclines, and fusidic acid were determined. Nineteen (9.3%) isolates were identified as MRS, and the presence of mecA in these isolates was confirmed by PCR. Virulence factors evaluation revealed that gelatinase was the most frequently detected (94.7%), followed by hemolysins (73.7%) and lipase (68.4%); 84.2% of the MRS isolates produced biofilm and icaA and icaD were detected in almost half of the MRS isolates (52.6%), but all were bap-negative. Resistance against other antimicrobial agents ranged from 0 (fusidic acid, ciprofloxacin, norfloxacin, enrofloxacin) to 100% (nalidixic acid). Resistance to nalidixic acid and nalidixic acid-tetracycline were the most common antimicrobial resistance profiles (31.6%). This study confirms that despite the low prevalence of MRS, isolates frequently express other virulence traits, especially biofilm, that may represent a serious challenge to clinicians.     

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.     

Increased milk levels of transforming growth factor-alpha, beta1, and beta2 during Escherichia coli-induced mastitis

Among the gram-negative bacteria that cause mastitis, Escherichia coli are the most prevalent. The innate immune system provides initial protection against E. coli infection by detecting the presence of the foreign pathogens and by mounting an inflammatory response, the latter of which is mediated by cytokines such as IL-1β, IL-8, and tumor necrosis factor (TNF)-α. Although changes in these cytokines during mastitis have been well-described, it is believed that other mediators moderate mammary gland inflammatory responses as well. The growth factors/cytokines transforming growth factor (TGF)-α, TGF-β1, and TGF-β2 are all expressed in the mammary gland and have been implicated in regulating mammary gland development. In other tissues, these growth factors/cytokines have been shown to moderate inflammation. The objective of the current study was to determine whether TGF-α, TGF-β1, and TGF-β2 milk concentrations were altered during the course of E. coli-induced mastitis. The contralateral quarters of 11 midlactating Holstein cows were challenged with either saline or 72 cfu of E. coli, and milk samples were collected. Basal milk levels of TGF-α, TGF-β1, and TGF-β2 were 98.81 ± 22.69 pg/mL, 3.35 ± 0.49 ng/mL, and 22.36 ± 3.78 ng/mL, respectively. Analysis of whey samples derived from E. coli-infected quarters revealed an increase in milk levels of TGF-α within 16 h of challenge, and these increases persisted for an additional 56 h. Elevated TGF-β1 and TGF-β2 milk concentrations were detected in E. coli-infected quarters 32 h after challenge, and these elevations were sustained throughout the study. Because TGF-α, TGF-β1, and TGF-β2 have been implicated in mediating inflammatory processes, their induction during mastitis is consistent with a role for these molecules in mediating mammary gland host innate immune responses to infection.     

Evaluating the in vitro susceptibility of bovine mastitis pathogens to a combination of kanamycin and cefalexin: Recommendations for a disk diffusion test

Cows suffering from bovine mastitis have markedly reduced milk production because of inflammation within the udder subsequent to infection and damage from bacterial toxins. Antibiotic treatment is commonly used as a preventative and therapeutic measure for bovine mastitis. The most common pathogens include Staphylococcus aureus, various streptococci (Streptococcus dysgalactiae, Streptococcus uberis), and coliforms (Escherichia coli), which can be contracted from other infected cows or from the environment. A combination of kanamycin and cefalexin (1:1.5 wt/wt) is currently used therapeutically in Europe for the treatment of bovine mastitis, although standardized methods for the in vitro determination of the susceptibility of target pathogens have not been developed. This study evaluates the appropriate broth microdilution testing criteria for kanamycin and cefalexin administered in combination and reports the development of a disk diffusion test. At a ratio of kanamycin:cefalexin relevant to that observed in milk postadministration (10:1 wt/wt), the minimum inhibitory concentrations were determined against 307 isolates of target mastitis pathogens (staphylococci, streptococci, and E. coli). Based on achievable concentrations in milk and the resulting distribution of minimum inhibitory concentrations, preliminary broth breakpoints for kanamycin/cefalexin (10:1 fixed ratio) of ≤8/0.8 μg/mL susceptible, 16/1.6 μg/mL intermediate, and ≥32/3.2 μg/mL resistant were applied to evaluated staphylococci, streptococci, and E. coli. Parallel testing by disk diffusion and resulting error-rate bounded analysis using a combined disk concentration of 30 μg of kanamycin and 15 μg of cefalexin resulted in the establishment of preliminary disk interpretive breakpoints of ≥20 mm susceptible, 18 to 19 mm intermediate, and ≤17 mm resistant for staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae only), and E. coli.