Evaluation of tandem repeats for MLVA typing of Streptococcus uberis isolated from bovine mastitis

BACKGROUND: Streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. A simple and efficient typing method would be helpful in identifying S.uberis sources, virulent strains and cow to cow transmission. The potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats) for S. uberis mastitis isolates genotyping was investigated. RESULTS: The genomic sequence of Streptococcus uberis (strain 0104J) was analyzed for potential variable number tandem repeats (VNTRs). Twenty-five tandem repeats were identified and amplified by PCR with DNA samples from 24 S. uberis strains. A set of seven TRs were found to be polymorphic and used for MLVA typing of 88 S. uberis isolates. A total of 82 MLVA types were obtained with 22 types among 26 strains isolated from the milk of mastitic cows belonging to our experimental herd, and 61 types for 62 epidemiologically unrelated strains, i.e. collected in different herds and areas. CONCLUSION: The MLVA method can be applied to S. uberis genotyping and constitutes an interesting complement to existing typing methods. This method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of S. uberis mastitis in dairy cows

Staphylococcus aureus lipoteichoic acid triggers inflammation in the lactating bovine mammary gland

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100 $\mu$g/quarter, and a subclinical inflammatory response at 10 $\mu$g/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100 $\mu$g). The pro-inflammatory cytokine IL-1$\beta$ was induced in milk, but there was very little if any TNF-$\alpha$ and no IFN-$\gamma$. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureus LTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus.

T helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland.

Infectious mastitis cuts down milk production profitability and is a major animal welfare problem. Bacteria-induced inflammation in the mammary gland (MG) is driven by innate immunity, but adaptive immunity can modulate the innate response. Several studies have shown that it is possible to elicit inflammation in the MG by sensitization to an antigen subsequently infused into the lumen of the gland. The objective of our study was to characterize the inflammation triggered in the MG of cows sensitized to ovalbumin, by identifying the cytokines and chemokines likely to play a part in the reaction. Among immunized cows, responders mobilized locally high numbers of leukocytes. An overexpression of the genes encoding IL-17a, IL-17F, IL-21, IL-22 and INF-γ was found in milk cell RNA extracts in the early phase of the inflammatory response. At the protein level, IL-17A was detected in milk as soon as the first sampling time (8 h post-challenge), and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h post-challenge. In mammary tissue from challenged quarters, overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22, IL-26 and IFN-γ was observed. Neutrophil-attracting chemokines (CXCL3 and CXCL8) were found in milk, and overexpressed transcripts of chemokines attracting lymphocytes and other mononuclear leukocytes (CXCL10, CCL2, CCL5, CCL20) were detected in mammary tissue. Expression of IL-17A, as revealed by immunohistochemistry, was located in epithelial cells, in leukocytes in the connective tissue and in association with the epithelium, and in migrated alveolar leukocytes of challenged quarters. Altogether, these results show that antigen-specific inflammation in the MG was characterized by the production of IL-17 and IFN-γ. The orientation of the inflammatory response induced by the antigen-specific response has the potential to strongly impact the outcome of bacterial infections of the MG

Concentrations of antibodies to ovalbumin following immunization.

<p>Data are presented as a function of the immunization group (A) or of the mammary ASR category following intramammary ovalbumin challenge (B). Antibodies were quantified in serum by ELISA before immunization (D0), on the day of first recall (D35/R1), on the day of second recall (D77/R2), two weeks later (D90), just before the intramammary challenge with ovalbumin (D104/IM) and eight days later (D112). Antibody concentrations did not differ significantly between the OVA+IFA and OVA+IFA+MAMP groups. Concentrations differed significantly between the high and low responder groups when indicated (*; Mann & Whitney test).</p

Experimental scheme.

<p>See M&M section for explanation. Green arrows indicate immunizations by subcutaneous injections of ovalbumin. Red arrows signal intramammary challenges with ovalbumin.</p

Correlations between milk cell concentrations (MCC) and concentrations of the chemoattractants and pro-inflammatory cytokines that were detected in the milk of all the responder cows.

<p>MCC 0–24 hpi: milk cell concentrations during the onset of the inflammatory response (0, 8, 12 and 24 h post-infusion). MCC 0–92 hpi: milk cell concentrations during the whole monitoring period. Correlations were calculated with the Bravais-Pearson test over two periods of time: at the onset of inflammation (0–24 hpi) and throughout the observation period (0–96 hpi).</p

Influx of leukocytes following the intramammary challenge of 21 cows with ovalbumin.

<p>A) Kinetics of cell concentrations in milk of unimmunized control cows, cows immunized with ovalbumin in incomplete Freund adjuvant, or cows immunized with ovalbumin in IFA supplemented with MAMP (MDP and LTA) (n = 7 per group); B) Individual values of milk cell concentrations at the peak of the cellular recruitment (3×7 cows); C) percentage of neutrophils (median values), determined by examination of cytospin slides stained with May-Grünwald-Giemsa, of the cows that reacted following immunization with ovalbumin in IFA (IFA; n = 4) or immunization with ovalbumin in IFA supplemented with MAMP (IFA+MAMP; n = 5); D) Kinetics of the individual values of the 9 immunized cows that reacted to ovalbumin challenge without distinction of groups.</p

Analysis by immunohistochemistry of representative sections of mammary tissue of ovalbumin-infused glands.

<p>A) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue (cow #4019) to antibody against N-terminal peptide of bovine IL-17A; B) Immunoreactivity of the mammary tissue of another cow (#1039) to the N-term antibody; C) Inhibition of labeling by Ab to N-terminal IL-17A peptide with the peptide antigen (cow #4019); D) Negative control with Ab to ovalbumin and second antibody conjugated to horseradish peroxidase; E, F) Immunoreactivity of the epithelial lining the alveoli and of cells in the connective tissue of cows #4019 and 1039 to the abcam antibody; G-H) Immunoreactivity of mammary tissue of cows #4019 and 1039 to the to the C-term and antibody. Scale bars indicate 25 µm.</p