Streptococcal Mastitis Research Articles

Evaluation of tRNA intergenic spacer length polymorphism analysis as a molecular method for species identification of streptococcal isolates from bovine mastitis

Sixty-nine bovine mastitis streptococci belonging to the species Str. agalactiae (n = 13), Str. dysgalactiae (n = 16), Str. canis (n = 22), Str. uberis (n = 20) and Str. parauberis (n = 4) and six reference strains of the five streptococcal species were examined for their tRNA gene intergenic length polymorphism (tDNA-ILP) fingerprint pattern. Epidemiologically unrelated isolates from bovine mastitis cases were selected by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). Their results were compared with those obtained from biochemical and serological studies and with those obtained by PCR-mediated identification amplifying species-specific gene segments of the five streptococcal species. According to the present results tDNA-ILP allowed a correct identification of all Str. agalactiae, Str. uberis and Str. parauberis strains investigated also including the reference strains of each species showing species-specific banding pattern. However, all Str. dysgalactiae ssp. dysgalactiae and all Str. canis strains appeared with an undistinguishable pattern which did not allow an identification of the species.

Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis

In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac + strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac + strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis.

Use of partial budgeting to determine the economic benefits of antibiotic treatment of chronic subclinical mastitis caused by Streptococcus uberis or Streptococcus dysgalactiae

The economic effect of lactational antibiotic treatment of chronic subclinical intramammary infections due to Streptococcus uberis or Streptococcus dysgalactiae was explored by means of partial budgeting. Effects at cow level and herd level were modelled, including prevention of clinical mastitis episodes and the prevention of transmission of infections. Input variables for our deterministic model were derived from literature or based on 2002/2003 dairy prices and farming conditions in The Netherlands. Sensitivity analysis was used to examine the effect of uncertainty around input variables or changes in price estimates. On farms where pathogen transmission was prevented through proper udder health management, 3-d antibiotic treatment during lactation resulted in an average net profit of euro+11.62 over no treatment while 8-d antibiotic treatment had an average negative net result of euro-21.83. Sensitivity analysis showed that profitability depends on the probability of treatment-induced cure, pathogen transmission rates, culling rate, retention pay-off, and costs of antibiotic treatment. Three-day antibiotic treatment of chronic subclinical streptococcal mastitis is economically profitable over a range of input values for cure probabilities, transmission rates and losses due to culling. In contrast, 8-d lactational treatment is only profitable for very valuable animals, on farms where the risk of pathogen transmission is high and/or when the farmer is likely to cull a high percentage of cows with subclinical mastitis. Because bacterial flora, cow characteristics and management differ widely between farms, the economic outcome of lactational treatment of chronic subclinical streptococcal mastitis may be highly farm-dependent.

Identification of multiple linear epitopes of the plasminogen activator A (PauA) of Streptococcus uberis with murine monoclonal antibodies

Streptococcus (S.) uberis is a common cause of mastitis in cattle. A protein (PauA) secreted by this bacterium is capable of activating plasminogen from sheep and cattle. The PauA first binds to bovine plasminogen (b-plg) to form a PauA-plasminogen complex that subsequently binds to and activates b-plg to form plasmin. We have identified several linear epitopes of PauA that are recognized by murine monoclonal antibodies to PauA. Two of the monoclonal antibodies which neutralized the enzymatic activity of PauA, EC3 and 2.22, recognized common linear peptide sequences with similar charge and spacing patterns. These neutralization epitopes are located in the predicted alpha-domain of the PauA molecule. Further, these same epitopes are in critical structure/function domains identified in other studies. These characterizations may facilitate the design of an efficacious vaccine for streptococcal mastitis in the dairy cow.

A GapC chimera retains the properties of the Streptococcus uberis wild-type GapC protein

The GapC products of Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis share considerable homology at the DNA and amino acid levels. The high similarity at the protein level suggests that one GapC protein might be used as a single antigen to protect dairy cows against infections with the contagious S. agalactiae and the environmental S. dysgalactiae and S. uberis strains. Despite their similarities, immunization with the S. dysgalactiae GapC did not protect dairy cows from a challenge with S. uberis, suggesting the presence of regions in GapC that are involved in species-specific protection. To produce a single antigen that can be used to protect against all streptococcal mastitis infections, we constructed a GapC chimeric protein using the S. uberis GapC product as the backbone followed by non-conserved peptide regions from the S. agalactiae and S. dysgalactiae GapC proteins. We report that the chimeric GapC protein retains the enzymatic activity of the S. uberis GapC protein. In addition, we fused the chimera to the OmpF and LipoF transport sequences of Escherichia coli and the GapC chimeras were present in membrane fractions of E. coli. These extracts could be the basis of an antigen preparation for use in mastitis vaccines.

The management and treatment of environmental streptococcal mastitis.

Environmental streptococci are responsible for at least one third of all cases of clinical mastitis, with the proportion varying widely between herds. Each dairy farmer should know the etiology of mastitis in the herd to allow for appropriate management. Control requires lowering the prevalence of infection, and includes shortening the duration of and preventing new infections. Prompt and aggressive treatment of clinical cases may achieve a high clinical and bactriologic rate of cure. Dry-cow treatment remains highly effective in eliminating infection. Prophylactic dry-cow treatment remains of substantial benefit in preventing new intramammary infections and clinical mastitis. An internal teat sealant has shown a comparable effect in uninfected cows in field studies. Management of exposure to environmental streptococci is essential and requires assessment of the risk of exposure (especially in bedding and other lying areas), reduction of teat-end contamination, and good hygienic milking practices. The key is in the quality of application of management.

Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression.

During bacterial infection of the bovine mammary gland, large numbers of leukocytes migrate into the udder, resulting in the establishment of a host response against the pathogen. Currently, the specific leukocyte populations mediating this immune response are not well defined. In the studies described here, we analyzed blood and milk from healthy cows and cows with naturally occurring mastitis to determine if distinct alphabeta and gammadelta T-lymphocyte subsets were involved in the response of the udder to a mastitis pathogen and if the type of mastitis pathogen influenced the subset composition of these responding leukocytes. Although blood samples from cows with confirmed staphylococcal and streptococcal mastitis were characterized by increased numbers of gammadelta T cells, the most dramatic changes in leukocyte distributions occurred in milk samples from these cows, with a 75% increase in alphabeta T-cell levels and a 100% increase in gammadelta T-cell levels relative to the levels in milk samples from healthy animals. Interestingly, the increase in alphabeta T-cell numbers observed in milk from cows with staphylococcal mastitis was primarily due to increased numbers of CD4(+) T cells, while the increase in alphabeta T-cell numbers observed in cows with streptococcal mastitis was due to a parallel increase in both CD4(+) and CD8(+) T-cell numbers. The increased numbers of gammadelta T cells in milk from cows with staphylococcal and streptococcal mastitis were due to a selective recruitment of a distinct gammadelta T-cell subset (GD3.1(+)), while no change in the numbers of GD197(+) gammadelta T cells was observed. We also analyzed adhesion protein expression on blood and milk leukocytes and found that, in comparison to the situation for healthy cows, L-selectin was down-regulated and CD18 was up-regulated on leukocytes from cows with mastitis. Thus, shedding of L-selectin and up-regulation of CD18 by neutrophils may provide a sensitive indicator of early inflammatory responses during bovine mastitis. Overall, these studies suggest that distinct alphabeta and gammadelta T-cell subsets are involved in the host defense of the udder against mastitis infection and that selective recruitment of these T-cell subsets depends on the infectious agent involved.

Bovine Mastitis in Finland in 1988 and 1995 - Changes in Prevalence and Antimicrobial Resistance

Two surveys were carried out (during 1988 and 1995) to estimate the prevalence of bovine mastitis in Finland. In 1988, 17,111 quarter milk samples were obtained from 4495 cows, and in 1995 the corresponding figures were 10,410 and 2648. Antimicrobial susceptibility of mastitis pathogens was studied. Prevalence of mastitis on cow basis decreased from 47.8% in 1988 to 37.8% in 1995. Staphylococci was the largest group of pathogens isolated. The proportion of Staphylococcus aureus decreased and that of coagulase-negative staphylococci (CNS) increased. The proportion of strains resistant to at least one antibacterial drug increased with regard to S. aureus from 36.9% in 1988, to 63.6% in 1995 and with CNS from 26.6% to 49.7%. Most of the increase in antibacterial resistance was due to a higher number of beta-lactamase producing strains. Multiresistance also increased, but it was proportional to the overall increase in resistance. All the predominant mastitis streptococci were susceptible to beta-lactams tested.

Efficacy of parenteral administration of three antimicrobial agents in treatment of clinical mastitis in lactating cows: 487 cases (1989-1995)

Objective To evaluate the efficacy of parenteral administration of procaine penicillin G, spiramycin, or enrofloxacin in the treatment of clinical mastitis in lactating cows. Design Noncontrolled, clinical retrospective study. Animals 487 cows with mastitis involving 543 quarters. Procedure Clinical signs, histories, and results of bacteriologic examination, somatic cell count, and N-acetyl-β-d-glucosaminidase activity of milk samples taken before and 3 to 4 weeks after treatment were retrieved from hospital records. Cows treated parenterally with procaine penicillin G, spiramycin, or enrofloxacin for 3 to 5 days were included. Supportive treatment alone was given to 35 cows infected with Escherichia coli. Factors possibly affecting outcome were analyzed, using ANOVA, correlation analyses, and the Mann-Whitney test. χ2 Test was used to compare bacteriologic cure rates. Results Bacteriologic cure rates for mastitis caused by Staphylococcus aureus, coagulase-negative staphylococci, and streptococci were 34, 76, and 65%, respectively. Cure rates in cows in their first lactation and infected with S aureus and coagulase-negative staphylococci were significantly higher than those for older cows. In cows with mastitis caused by E coli, the cure rate was 74% for those treated with penicillin G and 71% for those not treated with antimicrobials. High N-acetyl-β-d-glucosaminidase activity in milk samples obtained at initial examination indicated a poor outcome in S aureus and streptococcal mastitis. Cows infected in the early lactation period had more severe inflammatory responses and clinical signs if infected with coagulase-negative staphylococci and coliforms. Clinical Implications 3 to 5 days of treatment with parenterally administered penicillin G for clinical mastitis caused by penicillin-susceptible S aureus strains is efficacious in young cows. Parenteral administration of spiramycin or enrofloxacin does not give satisfactory results in mastitis caused by penicillin-resistant S aureus. Use of antimicrobials in the treatment of mastitis caused by coliform bacteria is questionable. (J Am Vet Med Assoc 1998; 212:407-412)

Use of Modified Rambach Agar to Differentiate Streptococcus uberis from Other Mastitis Streptococci

The medium of Rambach was modified to permit differentiation of mastitis streptococci. A total of 377 streptococci isolated from bovine IMI was used in the study. Of the 159 strains identified as Streptococcus uberis, 151 strains (94.9%) were β-galactosidase-positive and yielded blue colonies on modified Rambach agar. In comparison, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus saccharolyticus, or Enterococcus faecalis strains were negative for propylene glycol utilization (red colonies) or β-galactosidase production; all strains yielded colorless colonies on modified Rambach agar. However, 5 of 35 (14.3%) Streptococcus equinus strains were also positive for β-galactosidase. Results indicate that modified Rambach agar is a convenient medium for the differentiation of the Strep. uberis from the other mastitis streptococci. Furthermore, modified Rambach agar could be easily incorporated as a screening medium for Strep. uberis in mastitis bacteriology laboratories.

Encapsulation of streptococci isolated from bovine milk.

Capsule expression was assessed by India ink staining in 95 streptococcal and enterococcal isolates representing 8 species. Organisms were evaluated after primary culture on blood agar and after storage in 10% skim milk at -80 degrees C. Seventeen of 95 primary culture samples were positive for an unstained halo (capsule) by India ink. Only 3 isolates were positive for capsule after being maintained for an extended period of time. To confirm results of India ink tests, an unencapsulated and encapsulated strain of each species was observed by electron microscopy. Cells of encapsulated strains were surrounded by thread-like fibers indicative of capsule, whereas cells of unencapsulated strains had a smooth surface. The importance of capsule in the pathogenesis of streptococcal mastitis is unknown. Studies on capsule expression by streptococci may facilitate investigations on the pathogenesis of streptococcal mastitis.

Mastitis Prevalence in Primigravid Heifers at Parturition

Prevalence of intramammary infection was determined for 382 primigravid heifers within 3 d postpartum on 11 Vermont dairy farms. Data collected during a 5-yr period are summarized. Duplicate quarter milk samples were cultured on tryptose-blood agar plates containing .1% esculin. Intramammary infections were diagnosed in 45.5% of the heifers and 18.7% of quarters. Staphylococcus species were the most prevalent bacteria isolated: they appeared in 25.4% of the heifers and 12.1% of quarters. Only 2.6% of the heifers were diagnosed with Staphylococcus aureus; 22.8% had udder infection caused by other staphylococcal species. Environmental mastitis pathogens, coliforms, and streptococci other than Streptococcus agalactiae were isolated from 14.9% of the heifers and 4.8% of quarters. The prevalence of mastitis among these primigravid heifers at parturition indicates a need to improve methods of diagnosis and control programs.

A selective medium for mastitis streptococci in milk

Since the introduction by Milk Marketing Boards throughout the UK of automated systems which enable total bacterial counts to be carried out economically and at frequent intervals on bulk milk consignments, streptococci associated with subclinical mastitis are more readily detected. In many cases the presence of these organisms has resulted in heavy financial penalties being imposed. We describe an improved selective medium which could assist in the rapid detection of such cases and allow prompt remedial action to be taken.

The effect of udder infection on the bacterial flora of the bulk milk of ten dairy herds.

The significance of udder infection as a factor increasing the bacterial count of herd bulk milk was measured monthly for one year in ten dairy herds in Southern England. Staphylococcus aureus or mastitis streptococci were detected in 86% of samples, usually in numbers between 1000 and 10 000 c.f.u./ml of milk. However, in 8 and 2% of samples respectively greater than 20 000 or 100 000 c.f.u. of mastitis pathogens/ml of milk were detected. This occurred most commonly in the herds with a high incidence of Streptococcus uberis mastitis. The total bacterial counts of the herds' milks varied between 13 960 and 46 230 c.f.u./ml in the winter and between 6510 and 63 000 c.f.u./ml in the summer. No correlation was found between bacteriological quality of herd milk and the cleanliness of the milking machine and pipeline as assessed by plant rinses.

A Freeze-Etching Study on Experimental Murine Mastitis

Streptococcal mastitis was produced experimentally in mice inoculated by the intramammary route; freeze-etched preparations from the affected mammary glands were studied by electron microscopy. The inoculated cocci were seen free in the acinar lumen, within luminal phagocytes and within cells of the epithelium. No significant pathological changes were noted in the junctional complexes between secretory epithelial cells. The results were comparable to those obtained by ultrathin sectioning and indicated that, while cocci can transfer from the acinar lumen into the substance of the epithelium and towards a subepithelial location, the junctional complexes between epithelial cells present a potential barrier to movement through the intercellular spaces.

Comparative Grouping of Mastitis Streptococci by Means of Co-Agglutination and Precipitation

Two hundred bovine mastitis streptococcal strains belonging to groups B, C, E, G and L were tested comparatively by means of the co-agglutination and precipitation technique. Identical results were obtained with the two tests in 191, or 95.5 %, of the strains. Six strains, which could not be grouped by co-agglutination, proved to belong to group B when grouped by precipitation. On the other hand, one strain which proved to belong to group G and two strains to group L when grouped by co-agglutination, could not be grouped by precipitation. Some cross-reactivity was observed between groups A and C, B and G, B and L. Only a few L strains showed marked cross-reactivity which was not easily distinguished from specific reactions. However, the cross-reactivity ought to be eliminated by dilution or adsorption. Using the precipitation test as a supplementary method, the easy and rapid co-agglutination test was found to be a suitable procedure for routine grouping of mastitis streptococci.

CAMP factor of group B streptococci: production, assay, and neutralization by sera from immunized rabbits and experimentally infected cows.

Quantitative assay methods for CAMP factor activity of group B streptococci and for neutralization of this activity by animal sera are described. The course of the CAMP reaction is influenced by preincubation of red cells with staphylococcal beta-hemolysin and the rate of the reaction by the concentration of CAMP factor. The production of CAMP factor is enhanced by substitution of maltose for glucose in culture broth. Rising inhibitory titers to CAMP activity in sera of rabbits injected with sterile culture supernates of group B streptococci suggest that CAMP factor is an antigen in the rabbit. Cows with experimentally induced group B streptococcal mastitis also demonstrated significant rises in neutralizing titers for CAMP activity.

The diagnosis of staphylococcal mastitis, with special reference to the characteristics of mastitis staphylococci.

It is now recognized that staphylococci may cause either an acute or chronic type of bovine mastitis. In some herds they may produce a greater incidence of infection than Streptococcus agalactice, and they are often of special concern in herds in which streptococcal mastitis has been eliminated. However, there is still lack of information as to the most suitable tests or criteria for the diagnosis of staphylococcal mastitis. More information is also needed regarding the characteristics of the staphylococci causing infection in the bovine udder.

448. Machine milking rate and mastitis

It is generally accepted that the udder possesses two main defence mechanisms against mastitis infections. The teat sphincter acts as a barrier against the entry of the pathogenic frequently present on the skin of the teats, and internal factors prevent the bacteria which gain access from setting up an infection. In recent years studies have been made on the relative importance of these two mechanisms, and particular attention has been paid to the relation of teat patency or ease of milking with the incidence of disease. Little (1) stressed the importance of the intact sphincter as a barrier, and suggested that the sphincters of older cows relax, making them more susceptible to infection. In an attempt to overcome the disadvantages of the manual assessment of teat patency, Espe & Cannon (2) used a special instrument. The results were not wholly satisfactory and no correlation with mastitis was found. More recently, Murphy (3) manually examined the teats of at least eighty-seven cows and found a trend towards a higher incidence of infection in quarters with patent teats. This latter author did not agree that relaxing of the teat sphincters with advance in age could account for the increased susceptibility of older cows to streptococcal mastitis (4). While Murnane (5) could not confirm Murphy's results, McEwan & Cooper (6), again using the manual method, obtained further evidence definitely suggesting that easy milking cows were more prone to mastitis. Although Stuart & Lancaster (7) found evidence confirming the importance of the teat orifice, they concluded that it was of less importance than those factors operating within the udder.

Studies on experimental streptococcal mastitis: IV. The Disease caused by Inoculation of Streptococcus agalactiae Strain S 13 into the Teat Canal of Goats

Summary It was found possible to control the speed of reaction to infection of goats inoculated into the teat canal with living strain S 13 streptococci. The speed of reaction apparently depended on the number of living organisms inoculated, and a delayed reaction followed inoculation of about 1,000 living organisms contained in 3·0 ml. of saline and 7·0 ml. of Todd-Hewitt broth. The course of the disease in goats was studied up to a period of 11 weeks after inoculation. No indirect test was completely reliable in detecting infection, but that found most sensitive was the leucocyte count in milk. Bacteriological examination after death of experimentally infected goats revealed the presence of Str. agalactiae in the supramammary lymphatic glands of inoculated sides of udders in four out of 13 cases. In three of the four positive cases the animal had an abnormally high temperature when destroyed. A dark colony variant of the strain of Str. agalactiae inoculated into the teat canal of goats was isolated in considerable numbers from the milk of two animals. Examinations of the udder and tests of the milk over a period of 11 weeks, failed to reveal any essential difference between the experimental condition caused by inoculation of Str. agalactiae into the teat canal of goats and naturally occurring bovine streptococcal mastitis as described in the literature. It is suggested, therefore, that this experimental condition in goats could be used for the elucidation of many problems concerned with bovine streptococcal mastitis.