Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132^T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes.Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132^T (=DSM 111922^T, = CCOS 1972^T).     

The early pathogenesis of bovine mastitis due to Escherichia coli

The pathogenesis of coliform mastitis was studied after infusing each of ten lactating quarters of three dairy cows with a large dose (ca. 1 x 10(9) colony-forming units) of virulent Escherichia coli strain B117. This approach was adopted first to maximize the chance of observing microscopic lesions in the tissues of a gland and secondly to overwhelm the differences that might be shown between animals in their response to the infection. The infected glands were examined at intervals of up to 4 h after infection by scanning and transmission electron microscopy and by light microscopy. The earliest lesions were seen after 1 h and consisted of necrosis and sloughing of the epithelial cells of the teat and lactiferous sinuses. After 2 h this was more severe, and was followed by an intense neutrophil response. Neutrophils migrated through the epithelial lesions and at first remained attached to the epithelial surface, forming large mounds. This resulted in gross underestimation of the number of cells in the lumen of the gland when neutrophils in the secretion were counted. At no stage was there evidence of attachment of organisms to the epithelial cells. Tissue damage did not extent beyond the basement membrane, which helps to explain the rapid clinical resolution seen in most field cases of the disease. There was considerable variation in the degree of response shown by the three cows, and also within the infected glands, where the damage was most severe in the lactiferous and teat sinuses. It seems unlikely that all aspects of the disease could be attributed to endotoxin.     

Diversity of yeasts from bovine mastitis in Southern Brazil

Mastitis is one of the most serious problems in the dairy cattle farms. The great majority of the cases are caused by bacteria, but lately there have been an increasing number of reports about cases of mycotic etiology. The objective of this work was to characterize the yeasts and yeast-like fungi associated with milk of cows with mastitis. Milk samples (n = 248) from a dairy belt situated around the region of Passo Fundo, hinterland of the state of Rio Grande do Sul, Southern Brazil, were analyzed. Aliquots of 0.1 ml of milk were inoculated on yeast extract-malta agar with chloramphenicol. After a period of incubation of 3-5 days at 22-25 degrees C, the counting of the morphologically distinct colonies was performed, as well as the isolation and identification through phenotypical and physiological criteria. It was possible to isolate 68 yeast species from 43 (17.3%) of the samples. The most frequent genera were Candida (37.9%), Pichia (19.1%), Cryptococcus (10.3%) and Rhodotorula (10.3%).     

Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S.

Dairy cattle mastitis has long been one of the most common and costly diseases in the dairy industry worldwide, due to its significant impact on milk production and animal welfare. Among all mastitis causing bacterial pathogens, Klebsiella pneumoniae causes the largest milk loss. To better understand the genomic features of this population, 180 K. pneumoniae strains isolated from dairy cattle mastitis milk in 11 U.S. states were sequenced. The phylogenetic analysis classified all mastitis-causing K. pneumoniae into two major phylogroups, with exclusive predominance in phylogroup KpI. Analysis of more than 61 sequence types, 51 capsular types and 12 lipopolysaccharide O-antigen types revealed great genomic diversity of this K. pneumoniae population. Approximately 100 gene units in accessory genomes were detected with significantly higher prevalence in bovine mastitis strains, compared to human-sourced or dairy environmental strains. The most notable genes were identified associated with ferric citrate uptake, lactose fermentation and resistance to heavy metals. The acquired antimicrobial resistance genes were identified in sporadic mastitis strains. This comprehensive genomic epidemiological study provides insights for a better understanding of the virulence of mastitis-causing K. pneumoniae strains and may lead to the development of novel diagnostic tools and preventive strategies.     

Plasmid profiles of Staphylococcus aureus causing bovine mastitis

A. B aumgartner , J. N icolet & M. E ggimann 1984. Plasmid profiles of Staphylococcus aureus causing bovine mastitis. Journal of Applied Bacteriology 56 , 159–163.
Plasmid profiles of Staphylococcus aureus , isolated from herds affected with chronic bovine mastitis, are heterogeneous, as shown by the study of 85 strains from 18 different farms. The strains isolated within a herd sometimes show related plasmid patterns, although even strains isolated from different quarters of the same udder may show variations in their plasmid content. Strains without antibiotic resistance are frequently free of plasmids. Therefore, the existence of mastitis Staph. aureus virulence plasmids is unlikely. Staphylococcus aureus , resistant to penicillin and streptomycin from 9 different farms show related plasmid patterns. This result confirms a geographic spread of certain mastitis Staph. aureus strains.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D4 (LTD4) or leukotriene C4 (LTC4) resulted in concentration dependent increases in prostacyclin release measured as 6-keto-prostaglandin F1 alpha. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment, with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B4 and leukotriene E4 (LTE4) were inactive at concentrations up to 10 microM. The induction of prostacyclin synthesis by LTC4 and LTD4 was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23187 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.     

Milk chloride level as an indicator of bovine mastitis

The aim of this study was to determine an influence of udder infection on milk chloride level and on milk productivity of cows of black and white race. Bacteriological analysis was performed by bacterial isolation from milk collected in sterile conditions from single lobes of mammary gland. The study was aimed to detect the following bacteria: Streptococcus agalactiae, Streptococcus uberis, Streptococcus agalactiae, Staphylococcus aureus, Staphylococcus epidermidis, Actinomyces pyogenes, Enterobacteriaceae rods, Corynebacterium bovis, and Micrococcus sp. Milk chloride level was determined by burette method in 1250 milk samples collected from entire udder. Milk productivity was determined on the day of bacterial isolation. Statistical analysis of the results of the study on udder infection, milk chloride level, and milk productivity of mammary gland did reveal lack of a simple correlations between those parameters and in the indirect manner indicated an influence of inappropriate maintenance conditions of tested cows on the health condition of their udder. It seems possible that alkalosis and acidosis in cows, taken into consideration in discussion section, could constitute a factor influencing the frequency of mastitis incidence.     

Identification and antibiogram of microbes associated with bovine mastitis

An investigation of Mastitis in cattle was carried out in Anand city and in nearby villages of Gujarat state using California Mastitis Test (CMT) kit. The prevalence of clinical and subclinical mastitis was found to be 5.5% and 15.75%, respectively. Staphylococcus aureus was identified through strain specific polymerase chain reaction; the remaining isolates identified on the basis of molecular analysis by 16S rDNA sequencing and phylogenetic analysis were Staphylococcus species, B. pumilus, Staphylococcus chromogenes, Bacillus species, and Pseudomonas species. In vitro antimicrobial susceptibility pattern of all the isolates was checked against 13 different antibiotics using the agar disc diffusion method. Highest bacterial resistance was observed with penicillin G and oxacillin antibiotics. It was also observed that the patterns of bacterial resistance have not changed in India over the years. The data supports the decrease in the incidence of mastitis but the rate of decrease is minimal. More effective control strategies are required.     

Immunorelevant proteins for the diagnosis of bovine staphylococcal mastitis

Staphylococcus aureus is a leading cause of bovine mastitis, a condition in which the udder of the cow is inflamed, reducing the quality and quantity of milk produced. Staphylococcal mastitis is a common infection that can develop into a chronic form. The segregation of infected animals is an important preventive practice but relies on an effective diagnostic method. For this purpose, we constructed a genomic library of S. aureus, and a screening step was conducted with antiserum produced using the total protein extract of the pathogen. The nucleotide sequences of the immunoselected clones were aligned with the genome of bovine S. aureus RF122, which enabled the identification of 65 different loci, including proteins related to metabolism, adhesion and cell wall production, toxins, regulatory proteins, and hypothetical proteins. The subcellular location of the immunoreactive polypeptides was also determined. Fifty-two percent were cytoplasmic, 34 % were located in areas exposed to the host’s immune system, and for 14 %, the location could not be determined. In silico analysis of the presence of these proteins in mastitis pathogens showed that Fib, ClfA, and the hypothetical protein SAB0166 were the only proteins specific for S. aureus. Therefore, these proteins are promising candidates for the serodiagnosis of staphylococcal mastitis.     

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.     

Inhibition of renin release following left circumflex bradykinin injection in dogs

We examined the renin secretory response to bradykinin (BK) injection into the left circumflex coronary artery (LCx) in dogs. Studies were conducted in anesthetized, carotid sinus denervated dogs which had been maintained on a low sodium diet. A 25 ga needle was inserted into the LCx for injection of BK (0.15 micrograms/kg). The rate of renin secretion (RS) was obtained during a 30 min control period, at 5 min after a non-hypotensive hemorrhage (10 ml/kg), at 1, 3 and 5 min after BK injection and at 15 min after the reinfusion of withdrawn blood. Four series of studies were conducted. Series I: BK injection into the LCx, Series II: saline injection into the LCx (sham), Series III: intravenous injection of BK, and Series IV: BK injection into the LCx in dogs with prior renal denervation. RS was suppressed by 80% (P less than 0.05) 5 min after injection of BK into the LCx. Saline injection (sham) into the LCx or intravenous BK administration did not inhibit RS. Furthermore, suppression of RS was not present in dogs with prior renal denervation. These results indicate that BK injection into the LCx causes a prompt reduction in the rate of RS and that this response is reflexively mediated by the renal nerves.     

Intra-pancreatic release of bradykinin during the course experimental pancreatitis in rat

Kallikrein/Kininogn activation is an important pathophysiological event in acute pancreatitis, leading to microcirculatory changes within the gland. Hitherto, only indirect measurements of pancreatic bradykinin formation have been performed, monitoring the peptide in the circulation and in the peritoneal exudate. In the present study, intra-pancreatic bradykinin release was assessed using microdialysis during experimental acute pancreatitis in rat. In mild, oedematous pancreatitis, induced by caerulein hyperstimulation, the levels of bradykinin within the gland were not elevated compared with those of control rats. However, in necrotic pancreatitis, induced by retrograde injection of taurocholate into the pancreatic duct, significantly elevated levels of intraglandular bradykinin were seen. Several rats in this group died whilst in a state of circulatory shock.     

Characterization of Staphylococcus aureus strains involved in human and bovine mastitis

Staphylococcus aureus is one of the main etiological agents of mastitis in different mammalian species. At present, it is unknown whether strains isolated from human mastitis cases share phenotypic properties and genetic background with those obtained from animal mastitis cases. Therefore, the objective of this study was to characterize S. aureus strains isolated from women with lactational mastitis and to compare them with the strains responsible for bovine mastitis and noninfectious strains. All the strains were genotyped by both pulsed field gel electrophoresis and multilocus sequence typing and submitted to a characterization scheme that included diverse assays related to pathogenic potential and antibiotic resistance. Apart from siderophore production, no significant association was observed between the strains from bovine and human mastitis. Statistical differences between human- and bovine-mastitis-associated strains were detected for some traits and virulence determinants, such as the presence of prophages and cna and hlb genes, which were more frequently found within the bovine group. On the contrary, resistance to penicillin was significantly higher among strains isolated from human lactational mastitis, probably related to the common presence of the blaZ gene. A high genetic diversity was found among the strains involved in mastitis in breastfeeding women.     

Distribution of virulence-associated genes in Streptococcus uberis isolated from bovine mastitis

Streptococcus uberis is an important pathogen that has been implicated in bovine mastitis but the virulence factors associated with pathogenesis are not well understood. The aim of this work was to examine 11 putative and known virulence-associated genes by PCR in 78 S. uberis strains isolated from infected animals in Argentina. Additionally, the distribution of virulence patterns over various herds was determined. Not all genes were present in the strains but all of the detected virulence-associated genes were present in combination. Forty-seven (60.3%) isolates carried seven to 10 virulence-associated genes. Further analysis revealed 58 virulence patterns. Different patterns were found within the same herd and among herds, demonstrating that strains with different virulence patterns were able to cause mastitis. Despite the large number of strains with different virulence patterns, strains with identical patterns was found. Detection of virulence-associated genes in individual S. uberis strains isolated from infected animals revealed one to 10 virulence genes. This may indicate that other virulence factors could be involved. The present study reveals the occurrence and distribution of 11 virulence-associated genes among S. uberis isolates from bovine mastitis in various herds and contributes to a better understanding of the pathogenicity of this bacterium.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Association of the M blood group system with bovine mastitis

Associations of the 11 bovine blood group systems with mastitis were examined in Red Danish dairy cattle. The mastitis status was followed during three lactational periods. A significant effect of the M blood group system on mastitis incidence was observed in the first and second lactation periods and a lower frequency of mastitis is found among animals lacking the M' factor as compared to those having the M' blood group factor. The significance of these results are discussed in view of the close relation between the M blood group system and the bovine lymphocyte antigens (BoLA), and the expected effect of eliminating the M' gene from the breed is estimated. Among the remaining 10 blood group systems, the T' system was the only system showing an overall effect on mastitis, and only in first and third lactation. However, the T' system was inconsistent with regard to the effect of the T' gene on the various mastitis diagnoses.     

Effect of central injection of bradykinin and bradykinin potentiating factor upon release of anterior pituitary hormones in ovariectomized female rats

The effects of third ventricular (IVT) injection of 25 μg of bradykinin (BK) upon plasma levels of LH, FSH, TSH, GH and prolactin were investigated in conscious ovariectomized female rats bearing indwelling jugular cannulae. Some animals were pretreated with bradykinin potentiating factor (BPF). Intravenous administration of BK had no effect upon hormone levels. IVT injection of BK significantly depressed plasma prolactin levels at 15 and 30 min post-drug, with levels returning to control values by 60 min. Pretreatment of animals with BPF (75 μg/3 μl) prolonged the prolactin suppression induced by BK for up to two hours. Plasma LH, FSH, TSH and GH levels in BK-rats were not significantly different from those of saline-injected animals at any time point measured. Neither BPF alone nor in conjunction with BK had any effects upon plasma levels of TSH; however, BK plus BPF suppressed FSH concentrations at 75 min post-BPF, while BPF alone appeared to increase GH levels at 45 min. In vitro incubation of hemipituitaries with 0.083, 0.83 or 8.33 μg/ml BK had no effect upon the release of LH, TSH or prolactin compared to control values. However, the secretion of GH and FSH was suppressed by the lowest dose of BK tested. These results suggest that BK may play a physiological inhibitory role in the regulation of prolactin, which can be augmented by preventing its degradation, i.e. via BPF. The effect of the peptide seems to be mediated by the CNS since neither intravenous injection of BK nor in vitro incubation of pituitaries with the peptide modified prolactin release.