leukotoxin and Mastitis--Bovine

We identified 199 Staphylococcus aureus isolates from quarter milk samples of 1,289 dairy cattle between 2014 and 2018. About 66% of the isolates were resistant to at least 1 antimicrobial agent; the highest rate of resistance was to penicillin, followed by resistance to ampicillin, erythromycin, and sulfadimethoxine. We obtained 30 methicillin-resistant S. aureus (MRSA) strains from 6 farms in 3 provinces. The MRSA strains exhibited a significantly higher resistance rate to most of the tested antimicrobials than the oxacillin-susceptible strains. The MRSA strains represented 5 genotypes: ST72-t324-SCCmec IV (n = 14), ST30-t1752-SCCmec IV (n = 8), ST188-t189-SCCmec NT (n = 6), ST188-t2284-SCCmec NT (n = 1), and NT-NT-SCCmec IV (n = 1). One of the ST188 MRSA strains represented a novel staphylococcal protein A (spa) type (t2284). In addition, 7 of the 8 ST30 MRSA strains were Panton-Valentine leukocidin (PVL)-positive and carried various staphylococcal enterotoxin encoding genes. This is the first report of PVL-positive ST30 MRSA-t1752-SCCmec IV from bovine mastitis in Korea. All of ST72-t324-SCCmec IV MRSA strains carried staphylococcal enterotoxin and leukotoxin encoding genes. They were also sensitive to most of the tested non-β-lactam antimicrobials. In contrast, ST188-t189 MRSA strains were resistant to multiple antimicrobials and predominantly carried the leukotoxin encoding gene. Taken together, these findings may indicate that dairy cows could be a major source for spreading MRSA strains, and contaminated milk could be a vehicle for transmission. Suitable hygienic measures should be established in dairy farms and processing plants to limit the likelihood of introducing MRSA into the food chain.

Topics: Animals; Bacterial Toxins; Cattle; Enterotoxins; Exotoxins; Female; Leukocidins; Mastitis, Bovine; Methicillin-Resistant Staphylococcus aureus; Milk; Republic of Korea; Staphylococcal Infections

Leukotoxin M/F'-PV (LukM/F'-PV) produced by bovine mastitis causing Staphylococcus aureus structurally comprises three domains, the β-sandwich, rim and stem domain. The rim and stem domains interacting with target cell membrane lipid rafts contributes to the virulent trait of the toxin. In the present study, two facts were hypothesized that neutralization of these domains will ebb LukM/F'-PV leukotoxicity. Secondly, the neutralizing antibodies can improve the leukotoxin detection sensitivity in bovine mastitis milk samples. The in silico mapping of S. aureus LukM C-termini comprising these domains predicted seven linear B-cell antigenic epitopes. The immune response of C-terminal truncated recombinant peptides rCtM19 (19 kDa; near carboxy-terminal) having four epitopes and rCtM15 (15 kDa; C-terminal) with three epitopes were evaluated for their diagnostic and neutralization potential. Anti-rCtM19 and anti-rCtM15 antibodies with enhanced immunogenicity had the most striking outcome in IgG-ELISA for detecting native determinants of leukotoxin. For the obtained ELISA values, ROC curve inferred a cut-off score of >0.102 OD405. The assay sensitivity in the range of 90-96% along with 100% specificity and AUC of 0.93-0.98 categorized subclinical and clinical from healthy bovine milk samples. As observed through in vitro neutralization and LDH assays, C-terminus specific antibodies (1:42 titer) deactivating leukotoxicity abolished LukM from interacting with lipid bilayer and LukF for forming pores on bovine neutrophil membrane. As a proof of concept, it was proved that peptide antibodies can be a more specific serodiagnostic and passive therapeutic molecules.

Topics: Animals; Antibodies, Neutralizing; B-Lymphocytes; Cattle; Enzyme-Linked Immunosorbent Assay; Exotoxins; Female; In Vitro Techniques; Limit of Detection; Mastitis, Bovine; Staphylococcus aureus

Leukotoxin M/F'-Panton Valentine (LukM/F'-PV), a beta pore-forming toxin secreted by Staphylococcus aureus, is a major virulence factor involved in the pathogenesis of bovine mastitis. The present study was aimed to determine immunogenicity of two recombinant subunits of LukM/F'-PV, rLukM (MW 38 kDa) and rLukF (MW 39 kDa), develop and validate an indirect enzyme linked immunosorbent assay (ELISA) using polyclonal antibodies raised in rabbits, and evaluate applicability of the assay to diagnose clinical and subclinical bovine mastitis. Additionally, in vitro assays were conducted to determine abilities of antibodies to neutralize cytotoxicity of the native leukotoxin. A total of 87 bovine milk samples (healthy, subclinical and clinical mastitis) were evaluated for the presence of toxin determinants. Receiver-operator characteristic curve for the experimental ELISA values statistically interpreted a cut-off score of >0.109 OD405, with an assay specificity of 100% and sensitivity in the range of 80-87.5%. In addition, area under curve of 0.93-0.98 revealed the test was accurate in categorizing samples from infected and non-infected bovine. The rLukF IgG-ELISA was more sensitive than rLukM IgG-ELISA. Furthermore, it was evident from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) dye reduction, indirect immunofluorescence and lactate dehydrogenase assays that anti-rLukM/rLukF antibodies, with high neutralizing titers, inhibited in vitro leukotoxic activity and protected bovine neutrophil membrane integrity from cytotoxicity of native leukotoxin. The findings demonstrated that antibodies produced from recombinant subunits contribute to specific and sensitive immunodiagnosis and may also have the potential to provide passive therapeutic benefit in the management of bovine mastitis.

Topics: Animals; Cattle; Enzyme-Linked Immunosorbent Assay; Exotoxins; Female; Mastitis, Bovine; Recombinant Proteins; Reproducibility of Results; Sensitivity and Specificity; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Using different typing methods (MLST, spa-, SCCmec- and agr-typing), PFGE and DNA microarray-based chip analysis, we characterized 20 MRSA strains isolated from livestock and veterinarians. PFGE analysis after macrorestriction with EagI provided seven different band patterns, which could be grouped into four clusters. One cluster consisted of all MRSA ST398 strains isolated from pigs, calves, mastitis milk and two veterinarians. One strain of ST398 from a veterinarian and the two strains of ST1 and ST8 formed the three other clusters. Antimicrobial susceptibility testing showed that 15 of 20 strains were resistant to ampicillin, cefoxitin, clindamycin, erythromycin, oxacillin, penicillin and tetracycline. All strains were susceptible to rifampin and vancomycin, 19 were susceptible to ciprofloxacin and 18 were susceptible to sulphamethoxazole/trimethoprim. Genes encoding different enterotoxins, leukotoxins and haemolysins were found in certain strains.

Topics: Animals; Anti-Bacterial Agents; Bacterial Typing Techniques; Cattle; Cluster Analysis; Drug Resistance, Multiple, Bacterial; Enterotoxins; Exotoxins; Female; Genotype; Humans; Livestock; Mastitis, Bovine; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Milk; Oligonucleotide Array Sequence Analysis; Staphylococcal Infections; Swine; Switzerland; Veterinarians

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF'-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF'-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF'-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF'-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF'-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF'-PV positive or negative isolates. These results establish that LukM/LukF'-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF'-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

Topics: Animal Diseases; Animals; Antibodies, Bacterial; Cattle; Exotoxins; Female; Goat Diseases; Goats; Mastitis, Bovine; Sensitivity and Specificity; Sheep; Sheep Diseases; Species Specificity; Staphylococcal Infections; Staphylococcus aureus; Titrimetry; Virulence

Staphylococcus aureus is a major pathogen for cattle, causing various forms of subclinical and clinical mastitis. Two groups of virulence factors (leukotoxins and superantigens) are supposed to play an important role in the initiation and/or the exacerbation of this disease. In order to detect all known and putative members of leukotoxins and SAgs (superantigens), we tested secreted factors of different S. aureus isolates in flow cytometry-based assays. Isolates were sampled from 68 cows of different farms and cultured for 24h in vitro. Supernatants were then coincubated with purified polymorphonuclear granulocytes (PMN) or combinations of blood mononuclear cells (MNC) and PMN. Viable PMN and MNC were determined by quantitative flow cytometry. In addition, we recorded the proliferation-inducing potential of isolate supernatants for bovine MNC. Based on these criteria, the supernatants of S. aureus isolates fell in three groups. The first group (n=32), termed LT-SNs (leukotoxin-containing supernatants), killed purified granulocytes (neutrophils and eosinophils) in vitro. The second group of supernatants (n=20), termed SAg-SN (superantigen-containing supernatants), induced activation and proliferation of mononuclear cells (MNC) and, only in the presence of MNC, resulted in a selective depletion of neutrophils after 24h in vitro. The third group of supernatants (n=16) contained neither LTs or SAgs. Functionally, SAg-SNs behaved like purified staphylococcal enterotoxin A (SEA) or SEB tested in parallel. The absence of SAg-like activity in LT-SNs was confirmed by heat treatment of LT-SNs, which destroyed the leukocytotoxic activity, but did not reveal any MNC-activating potential. This study, therefore, suggests, that pathogenic S. aureus isolates either produce leukotoxins or superantigens and that both groups of virulence factors can easily be differentiated by the functional assays described. The prevalence of leukotoxin- or superantigen-producing isolates was comparable among cattle with subclinical (LT=41%; SAg=30.8%) mastitis. The higher frequency of LT-producing isolates in cases of clinical mastitis (LT=55.2%; SAg=27.6%) was not significant. At least, these findings argue against the dominant role of superantigens or leukotoxins in S. aureus-induced bovine mastitis.

Topics: Animals; Cattle; Dairying; Exotoxins; Female; Flow Cytometry; Leukocytes; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus; Superantigens; Virulence