Mastitis pathogens of cattle
In the sections which follow, the special features of each mastitis associated with one or a group of pathogens will be described using the usual format of the book. Mastitis in cattle is categorized as being associated with contagious, teat skin opportunistic or environmental pathogens, and as being common or less common. The features that are unique to the diagnosis, treatment and control of each mastitis pathogen will be outlined but details applicable to all causes of mastitis have been presented above.
Etiology Staphylococcus aureus is a major pathogen of the mammary gland and a common cause of contagious bovine mastitis. S. aureus also causes mastitis in sheep and goats
Epidemiology Major cause of mastitis in dairy herds without an effective mastitis control program. Prevalence of infection 50–100%; prevalence of 1–10% in herds with low bulk tank milk SCCs, 50% in high-SCC herds, quarter infection rate 10–25% in high-SCC herds. Source of infection is infected udder; infection transmitted at milking. Chronic or subclinical S. aureus mastitis is of major economic importance
• Chronic S. aureus mastitis is most common and is characterized by high SCC and gradual induration of udder, drop in milk yield and atrophy with occasional appearance of clots in milk or wateriness
• Acute and peracute S. aureus mastitis most common in early lactation. Acute swelling of gland with fever; milk is abnormal with thick clots and pus; gangrene of gland and teat in peracute form. Systemic reaction with anorexia, toxemia, fever, ruminal stasis
Clinical pathology Culture individual cow milk sample; indirect tests are high SCC and California Mastitis Test results
Necropsy findings Peracute, acute, and chronic (recurrent) clinical mastitis, subclinical mastitis common
• Lactating cows – cure rates for lactating cows with subacute staphylococcal mastitis less than 50%. Intramammary infusions daily for at least 3 days, preferably 5–8 days
• Peracute mastitis – antimicrobial agents parenterally and intramammary that are beta-lactamase-resistant, fluid and electrolyte therapy
• Dry cow therapy – chronic or subclinical mastitis best treated at drying off with long-acting intramammary antimicrobial infusions that are beta-lactamase-resistant
• Prevent new infections by early identification, culling infected cows and good milking procedures, including hygienic washing and drying of udders and teats before milking and postmilking germicidal teat dips. Regular milking machine maintenance. Consider segregation of infected cows
Coagulase-positive S. aureus is a major pathogen of the bovine mammary gland and a common cause of contagious mastitis in cattle. S. aureus also causes mastitis in sheep and goats.
Historically, S. aureus was one of the most common causes of bovine mastitis in dairy cattle worldwide. In the last 25 years, the prevalence of infection and the occurrence of clinical mastitis due to S. aureus has decreased in herds using effective mastitis control measures. However, surveys indicate that 50–100% of herds may be infected. In low-SCC herds, the prevalence of infection in cows ranges from 1–10%. In other herds, especially those with high SCCs, up to 50% of cows may be infected with S. aureus, with quarter infection rates ranging from 10–25%. The prevalence of infection of S. aureus in heifers at parturition can range from 5–15%. The majority of intramammary infections due to S. aureus are subclinical. The incidence of clinical mastitis due to S. aureus is dependent on its prevalence of infection in the herd. With an effective mastitis control program, the most common causes of clinical mastitis are the environmental pathogens. However, in some herds with a low rolling SCC, incidence of clinical mastitis due to S. aureus ranges from 190–240 cases/100 cows/year, with about 47% of the clinical cases being S. aureus.1
S. aureus is ubiquitous in the environment of dairy cattle. The infected mammary gland of lactating cows is the major reservoir and source of the organism.2 The prevalence of intramammary infection in primiparous heifers at parturition ranges from 2–50% and may represent an important reservoir of infection in herds with a low prevalence of infection. The organism may be present on the skin of the teats and external orifices of heifers, bedding materials, feedstuffs, housing materials, nonbovine animals on the farm, and equipment. In herds with a high prevalence of infection (> 10% of cows), the organism was present in bedding, the hands and noses of dairy herd workers, insects and water supplies.2 Transmission between cows occurs at the time of milking by contaminated milkers’ hands and teat cup liners. Although S. aureus can multiply on the surface of the skin and provide a source of infection for the udder, the teat skin lesions are usually infected originally from the udder, and teat skin is a minor source of infection.
The hornfly (Hameotobia irritans) is an important vector for transmitting S. aureus mastitis in heifers, particularly in herds with scabs on the teat ends of heifers.3,4 Prevention of high populations of flies in heifers is therefore needed to decrease new infections in this group.
Several animal risk factors influence the prevalence of infection and the occurrence of clinical mastitis due to S. aureus.
Abrasions of the teat orifice epithelium are an important risk factor for S. aureus mastitis.5 In experiments, teat canal infection or colonization may develop in 93% of experimentally abraded teat canal orifices compared to 53% in control quarters. Chapping of the teats and thickness of the teat barrel are correlated and significantly influence recovery of S. aureus from the skin.6
The presence of minor pathogens such as coagulase-negative staphylococci protects against new intramammary infections associated with the major pathogen S. aureus.7 This may be the result of an elevated SCC or an antimicrobial-like substance provided by the coagulase-negative staphylococci that inhibits the growth of S. aureus. Conversely, quarters infected with coagulase-negative staphylococci may be more susceptible to new infections with S. agalactiae. Quarters that are infected with C. bovis are protected against S. aureus infection but not protected against most streptococcal species.
The prevalence of intramammary infection and subclinical infection due to S. aureus increases with parity of the cow. This is probably due to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without a mastitis control program.
The presence of periparturient diseases such as dystocia, parturient paresis, retained placenta and ketosis has been identified as a risk factor for mastitis. The occurrence of sole ulcers in multiple digits may be associated with S. aureus in the first lactation.8
Experimentally, the presence of certain bovine lymphocyte antigens increased the susceptibility to S. aureus infection9 but heritability estimates of susceptibility after experimental challenge were low and unstable.
The infection rate of S. aureus is dependent on the ability of the immune system to recognize and to eliminate the bacteria.10 Staphylococcal antibodies are present in the blood of infected cows but they appear to afford little protection against mastitis associated with S. aureus. This may be due to the low titer of the antibodies in the milk. Antibody titers in the serum rise with age and after an attack of mastitis.
The development or persistence of S. aureus mastitis depends on the interaction between invading bacteria and the host’s defense system, principally the somatic cells in an infected gland, which are more than 95% polymorphonuclear cells. The number of bacteria isolated from milk samples of S. aureus-infected mammary glands is characterized by a cyclic increase and decrease concomitant with an inverse cycling of the SCC. This relationship between SCC and numbers of bacteria indicates that the cells within the mammary gland have a central role in the pathogenesis of S. aureus infection.11 There appear to be qualitative changes in the ability of the animal’s somatic cells to phagocytose the bacteria. During the period of high SCC, the cells are able to kill bacteria 9000 times more efficiently than during the low-SCC period. The relative inability of the polymorphonuclear cells to kill bacteria during the low-SCC period may explain the source of reinfection. Phagocytosis and killing of the bacteria may also be inefficient because of low concentrations of opsonins, a lack of energy source, and the presence of casein and fat globules in the milk. The function of the intramammary polymorphonuclear cell (somatic cells) may also be affected by immunosuppression induced by cortisol and dexamethasone in treated cows.12
Several herd-level management risk factors are important for the spread of S. aureus.13 Poor teat and udder cleaning can allow spread of the organism among quarters of the same cow, and can allow contamination of milking units, which are commonly transferred among cows without washing or rinsing. The use of high-line parlors is a risk; this may be due to the greater fluctuation in vacuum, especially when units are removed, leading to a greater occurrence of teat end impacts in which bacteria in the milking unit may enter the teat canal to establish a new udder infection.
Extensive surveys reveal that management procedures that are most effective in reducing infection rates and cell counts associated with infections with S. aureus are:
• Maintaining a good supply of dry bedding for housed cows
• Thorough disinfection of the teat orifice before infusing intramammary preparations
Failure to use these management techniques will increase the risk of intramammary infection with S. aureus.
S. aureus has several virulence factors that account for its pathogenicity and persistence in mammary tissue in spite of adequate defense mechanisms and antimicrobial therapy. Most isolates from cattle appear to be host-adapted and different from human S. aureus isolates. S. aureus has the ability to colonize the epithelium of the teat and the streak canal, and can adhere and bind to epithelial cells of the mammary gland. The specific binding is to the extracellular matrix proteins fibronectin and collagen, which can induce the epithelial cell to internalize the organism, protecting it from both exogenous and endogenous bactericidal factors. Some strains of S. aureus are capable of invading bovine mammary epithelial cells in culture, and the invasion process requires eukaryotic nucleic acid and protein synthesis as well as bacterial synthesis.14
Some strains of S. aureus produce toxins, some of which may cause phagocytic dysfunction. The beta toxin, or a combination of alpha toxins and beta toxins, is produced by most pathogenic strains isolated from cattle but its pathogenic significance is uncertain. The beta toxin damages bovine mammary secretory epithelial cells, increases the damaging effects of alpha toxin, increases the adherence of S. aureus to mammary epithelial cells and increases the proliferation of the organism.15 All strains produce coagulase (hence the term coagulase positive S. aureus), which converts fibrinogen into fibrin; this appears to assist the invasion of tissues. Leukocidin produced by S. aureus may inactivate neutrophils.
Many staphylococcal strains (coagulase-negative and -positive) are able to produce an extracellular exopolysaccharide layer surrounding the cell wall.16 This capsular structure and its production of slime have been associated with virulence against host defense mechanisms.
A major pathogenic factor is the ability of the organism to colonize and produce microabscesses in the mammary gland so that it is protected from normal defense mechanisms, including phagocytic activity from neutrophils. The difficulty in removing staphylococci from an infected quarter is due largely to the bacteria’s ability to survive in intracellular sites. There is also an ability to convert to a nonsusceptible L-form when exposed to antimicrobial agents, and to return to standard forms when the antimicrobial is withdrawn.
Phage typing and ribotyping can be used to classify strains from clinical and subclinical S. aureus mastitis.17
DNA fingerprinting techniques, using polymerase chain reaction, are also being used to differentiate various strains of the organism.18 A large number of different types of S. aureus can be isolated from cases of bovine mastitis but a few types predominate within different countries.19 Surveys have found that only a small number of genotypes cause most cases of S. aureus mastitis,18 which may be useful information in determining the dynamics of infection in a herd and how infection spreads from cow to cow. Fine-structure molecular epidemiological analysis of S. aureus recovered from cows in the USA and Ireland indicates that only a few specialized clones of S. aureus are responsible for the majority of cases of bovine mastitis, and that these clones have a broad geographical distribution.20 A predominant strain is usually responsible for most clinical and subclinical S. aureus infections in a herd,21,22 and it is currently believed that S. aureus is a clonal organism that spreads from cow to cow. Moreover, most strains isolated from milk are different from strains isolated from the teat skin. In other words, most S. aureus strains isolated from mastitis demonstrate both host and site specificity.22 This has important implications in the control of mastitis associated with S. aureus, as a rational and effective strategy for control of intramammary infections should be directed against clones that commonly cause disease.
The overall prevalence of mastitis due to S. aureus is much higher than for S. agalactiae, and the need for culling causes much greater economic consequences. The risk of new infections is of continuing concern. Response to treatment is comparatively poor, and satisfactory methods for the eradication of staphylococcal mastitis from infected herds have yet to be devised.
The presence of S. aureus in market milk may present a degree of risk to the consumer because of the organism’s capacity to produce enterotoxins and a toxic shock syndrome toxin, which cause serious food poisoning. Mastitic milk does not constitute any large risk for S. aureus enterotoxin food poisoning.23
The disease can be reproduced experimentally by the injection of S. aureus organisms into the udder of cattle and sheep but there is considerable variation in the type of mastitis produced. This does not seem to be due to differences in virulence of the strains used, although strain variations do occur, but may be related to the size of the inoculum used or, more probably, to the lactational status of the udder at the time of infection. It is possible to induce S. aureus infection in the bovine teat cistern; the teat tissues are able to mount a marked local inflammatory response but in spite of large numbers of neutrophils that invade the teat, they are unable to control the infection, except when the numbers of bacteria are low.24
Infection during early lactation may result in the peracute form of mastitis, with gangrene of the udder. During the later stages of lactation or during the dry period new infections are not usually accompanied by a systemic reaction but result in the chronic or acute forms. Chronic S. aureus mastitis in cows has been converted to the peracute, gangrenous form by the experimental production of systemic neutropenia.
In the gangrenous form the death of tissue is precipitated by thrombosis of veins causing local edema and congestion of the udder. S. aureus are the only bacteria that commonly cause this reaction in the udder of the cow, and the resulting toxemia is due to bacterial toxins and tissue destruction. Secondary invasion by E. coli and Clostridium spp. contributes to the severity of the lesion and production of gas.
The pathogenesis of acute and chronic S. aureus mastitis in the cow is the same, the variation occurring only in degree of involvement of mammary tissue. In both forms each focus commences with an acute stage characterized by proliferation of the bacteria in the collecting ducts and, to a lesser extent, in the alveoli. In acute mastitis the small ducts are quickly blocked by fibrin clots, leading to more severe involvement of the obstructed area.
In the chronic form there are fewer foci of inflammation and the reaction is milder; the inflammation is restricted to the epithelium of the ducts. This subsides within a few days and is replaced by connective tissue proliferations around the ducts, leading to their blockage and atrophy of the drained area. The leukocyte infiltration into the stroma, the epithelial lining and the lumina indicate an obvious deficiency of secretory and synthesizing capacity due to limitation of the alveolar lumina and the distension of the stroma area.
A characteristic of chronic S. aureus mastitis that is important in its diagnosis is the cyclical shedding of the bacteria from the affected quarter. Paralleling this variation is a cyclical rise and fall in the number of polymorphonuclear cells in the milk, and their capacity to phagocytose bacteria.11 In some cases abscesses develop and botryomycosis of the udder, in which granulomata develop containing Gram-positive cocci in an amorphous eosinophilic mass, is also seen.
The most important losses are caused by the chronic form or subclinical form of mastitis. Although 50% of cattle in a herd may be affected, only a few animals will have abnormalities recognizable by the milker. Many cases are characterized by a slowly developing induration and atrophy with the occasional appearance of clots in the milk or wateriness of the first streams. The SCC of the milk is increased, as well as the CMT results of infected quarters, but the disease may go unnoticed until much of the functional capacity of the gland is lost. The infection can persist and the disease may progress slowly over a period of many months.
Acute and peracute staphylococcal mastitis are rare but do occur and can be fatal, even if aggressively treated.
Acute S. aureus mastitis occurs most commonly in early lactation. There is severe swelling of the gland and the milk is purulent or contains many thick clots. Extensive fibrosis and severe loss of function always result.
Peracute S. aureus mastitis occurs usually in the first few days after calving and is highly fatal. There is a severe systemic reaction with elevation of the temperature to 41–42°C (106–107°F), rapid heart rate (100–120 beats/min), complete anorexia, profound depression, absence of ruminal movements and muscular weakness, often to the point of recumbency. The onset of the systemic and local reactions is sudden. The cow may be normal at one milking and recumbent and comatose at the next. The affected quarter is grossly swollen, hard and sore to touch, and causes severe lameness on the affected side.
Gangrene is a constant development and may be evident very early. A bluish discoloration develops that may eventually spread to involve the floor of the udder and the whole or part of the teat, or may be restricted to patches on the sides and floor of the udder. Within 24 hours the gangrenous areas become black and ooze serum and may be accompanied by subcutaneous emphysema and the formation of blisters. The secretion is reduced to a small amount of bloodstained serous fluid without odor, clots or flakes. Unaffected quarters in the same cow are often swollen, and there may be extensive subcutaneous edema in front of the udder caused by thrombosis of the mammary veins. Toxemia is profound and death usually occurs unless early, appropriate treatment is provided. Even with early treatment the quarter is invariably lost and the gangrenous areas slough. Separation begins after 6–7 days, but without interference the gangrenous part may remain attached for weeks. After separation, pus drains from the site for many more weeks before healing finally occurs.
Bacteriological culture of milk is the best method for identifying cows with S. aureus intramammary infection.25 A problem in the laboratory identification of S. aureus is that bacteria are shed cyclically from infected quarters, so that a series of samples are necessary to increase overall test sensitivity. The sensitivity of a single sample may be as low as 75%. Factors that have the greatest impact on the sensitivity of culture, in order of importance, are:
• The time interval between repeated milk sample collection strategies.26
Quarter samples taken on day 1 and repeated either on day 3 or 4, and cultured separately using 0.1 mL of milk for culture inoculum, were predicted to have sensitivities of 90–95% and 94–99%, respectively.26 Repeated quarter samples collected daily and cultured separately gave a sensitivity of 97% and a specificity from 97–100%.27 Culturing of composite milk samples instead of individual quarter samples increases the number of false-negative results in diagnosing S. aureus mastitis,28 but the sensitivity of composite samples can be increased by using 0.05 mL of milk for inoculation. Freezing of milk samples before processing either does not affect the bacterial count or enhances it by about 200%; the latter response is attributed to fracturing of cells containing viable S. aureus bacteria. Bacterial counts of more than 200 cfu/mL are commonly used as a criterion for a positive diagnosis of infection.
The culture of 0.3 mL of bulk tank milk for S. aureus using special Baird–Parker culture media is a practical method for detecting the organism in bulk tank milk and monitoring its spread in dairy herds;29 the sensitivity and specificity for detection of the bacteria ranged from 90–100%.
In an attempt to decrease the cost of sampling all quarters for culture, an alternative strategy is to use the SCC as a screening test in order to identify which cows to culture for S. aureus. For all intramammary infections, the sensitivity and specificity of SCC range from 15–40% and 92–99%, respectively. Composite milk sample SCCs have a low sensitivity, ranging from 31–54% for detecting cows with S. aureus.27 Individual quarter SCCs have a higher sensitivity, ranging from 71–95% depending on the study and cutpoint chosen, but quarter sampling is impractical as SCC is usually performed on a composite sample.30 Both composite and quarter milk SCC testing result in an unacceptably high proportion of infected cows being missed,27,30 and are therefore not currently recommended as a screening test if the goal is to identify all cows with a S. aureus intramammary infection in the herd.
The CMT has also been used as a screening test to identify quarters or cows to culture. Using a CMT trace, 1, 2, or 3 to indicate the presence of an intramammary infection produced a range of sensitivities from 0.47–0.96 and specificities of 0.41–0.80.30
In summary, culture of quarter milk samples (preferably) or a composite milk sample is superior to a quarter SCC or CMT for the diagnosis of S. aureus intramammary infection.30 Culture is strongly preferred if it is important to identify all positive cows in a herd because the sensitivity of indirect tests (such as SCC, CMT) is inadequate.
ELISA tests for detecting S. aureus antibody in milk have been developed25,31 but are not widely used. Rapid laboratory tests incorporating these ELISAs, including a Staph-zym test, have demonstrated 84–90% accuracy in identifying staphylococci.32
The acriflavine disk assay is a practical, accurate method for differentiating S. aureus isolates from non-S. aureus staphylococci.33
In peracute staphylococcal mastitis, the affected quarter is grossly swollen and may contain bloodstained milk dorsally but only serosanguineous fluid ventrally. There is extreme vascular engorgement and swelling, often progressing to moist gangrene of the overlying skin. Bacteria are not isolated from the bloodstream or tissues other than the mammary tissue and regional lymph nodes. Histologically there is coagulation necrosis of glandular tissue and thrombosis of veins.
In milder forms of staphylococcal mastitis the invading organisms often elicit a granulomatous response. Microscopically, such ‘botryomycotic’ cases are characterized by granulomas with a central bacterial colony and by progressive fibrosis of the quarter.
Because of the occurrence of the peracute form in the first few days after parturition, the intense depression and inability to rise, the dairy producer may conclude that the cow has parturient paresis, which is characterized by weakness, recumbency, hypothermia, rumen stasis, dilated pupils, tachycardia with weak heart sounds and a rapid response to intravenous calcium gluconate. The mammary gland is usually normal in parturient paresis.
Peracute S. aureus mastitis is characterized by marked tachycardia, fever, weakness and evidence of severe clinical mastitis with swelling, heat, abnormal milk with serum and blood, and sometimes gas in the teat and often with gangrene of the teat up to the base of the udder. Other bacterial types of mastitis, particularly E. coli and A. pyogenes, may cause severe systemic reactions but gangrene of the quarter is less common.
Peracute coliform mastitis is a much more common cause of severe mastitis than S. aureus mastitis. The chronic and acute forms of staphylococcal mastitis are indistinguishable clinically from many other bacterial types of mastitis and bacteriological examination is necessary for identification.
The bacteriological cure rates for the treatment of S. aureus mastitis with either intramammary infusion or parenteral antimicrobial administration are notoriously less than satisfactory, particularly in the lactating cow. Bacteriological cure rates after antimicrobial treatment seldom exceed 50% and infections commonly persist throughout the lifetime of the cow. There are three likely reasons: inadequate penetration of the antimicrobial agent to the site of infection, formation of L-forms of S. aureus, and beta-lactamase production.
There is inadequate penetration of the antimicrobial agent into the site of intramammary infection in the lactating cow and the organism survives in phagocytes that are inaccessible. There may also be inactivation of the antimicrobial by milk and serum constituents, and the formation of L-forms of the organism during treatment, varying between 0% and 80% of bacteria.
Antimicrobial resistant strains of S. aureus occur and are often beta-lactamase producers, the enzyme conferring resistance to beta-lactam antimicrobial agents such as penicillin G, penethamate, ampicillin and amoxicillin. Cloxacillin and nafcillin are effective, but only against Gram-positive bacteria; they are less effective against nonlactamase staphylococci. Clavulanic acid added to amoxicillin overcomes this beta-lactamase resistance. So does cloxacillin added to ampicillin, and this is made use of in a popular intramammary formulation. First- and third-generation cephalosporins and erythromycin are effective against beta-lactamase-producing staphylococci, and first- and third-generation cephalosporins are also effective against Gram-negative bacteria. A cephapirin dry cow product administered to heifers with S. aureus infections resulted in bacteriological cure and left the quarters clear well into their first lactation.34 Intramammary cloxacillin and ampicillin is generally considered to be the preferred initial treatment for S. aureus mastitis because beta-lactamase production by S. aureus is sufficiently common.
Antimicrobial therapy for S. aureus subclinical mastitis during the lactating period is not economically attractive because of low bacteriological cure rates, discarding of milk during the withholding period and the lack of an economically beneficial increase in production following treatment. Dry cow treatment at the end of lactation is much more effective, being successful in 40–70% of cases, although treatment should be attempted in heifers infected early in lactation. Cows that are infected with S. aureus should be appropriately identified, segregated if possible and milked last or with separate milking units.35 Culling of infected cows is also an option for consideration, but a detailed economic analysis of this popular recommendation is lacking.
The treatment of clinical cases of S. aureus mastitis using intramammary antimicrobial infusions is less than satisfactory but is often done. However, clinical recovery following therapy does not necessarily eliminate the infection and some of the published literature on cure rates has not made the distinction between clinical and bacteriological cure rates. In general, the cure rate depends on the duration of infection, the number of quarters infected, whether the strain of S. aureus is a beta-lactamase producer, the immune status of the cow, the antimicrobial agent administered and the duration of treatment. Current recommendations to ensure the best treatment success rate are to combine intramammary and parenteral antimicrobial treatment or use extended intramammary treatment for 4–8 days. Penicillin G is regarded as the antimicrobial agent of choice for S. aureus strains that are penicillin-sensitive.22
The following intramammary infusions, given daily at 24-hour intervals for three treatments (unless stated otherwise) have been used for the treatment of clinical cases of S. aureus mastitis, with expected clinical cure rates of about 30–60% in lactating cows. Subclinical cases are left until the cow is dried off:
• Sodium cloxacillin (200–600 mg for three infusions)
• Penicillin–streptomycin combination (100 000 units – 250 mg)
• Penicillin–tylosin combination (100 000 units – 240 mg)
• Novobiocin (250 mg per infusion for three infusions)
• Cephalosporins – most strains of S. aureus are sensitive to cephapirin36
• Pirlimycin-extended therapy (two 50 mg doses, 24 hours apart, then 36-hour withhold, then cycle repeated twice, equivalent to infusing at 0, 24, 60, 84, 120, and 144 hours).
In a study of 184 cases of subclinical S. aureus mastitis in New York, commercially available intramammary infusions were not significantly more effective than untreated controls (43% bacteriological cure), with the following bacteriological cure rates: erythromycin (65%), penicillin (65%), cloxacillin (47%), amoxicillin (43%), and cephapirin (43%).37
A slightly more effective treatment for subclinical S. aureus intramammary infection, with a cure rate of 50%, is simultaneous intramammary infusion of amoxicillin (62.5 mg) and intramuscular injection of procaine penicillin G (9000000 units).38 This study was the first to demonstrate that combined parenteral and intramammary therapy was more effective than intramammary infusion alone. Because of the persistence of the infection in each herd the final choice of the antimicrobial to be used should be based on a culture and susceptibility test; the latter is to determine whether the predominant S. aureus strain in the herd is a beta-lactamase producer; this is because beta-lactamase-producing strains are harder to cure and require a specific antibiotic protocol.22 The bacteriological cure rate for penicillin-sensitive infections treated with parenteral and intramammary penicillin G was 76%, compared to beta-lactamase-producing strains treated with parenteral and intramammary amoxicillin– clavulanic acid (29%).22
The application of cytokines as an adjunct to antimicrobial therapy may help to increase the number of phagocytes in the mammary gland and enhance cell function. The experimental intramammary infusion of recombinant interleukin into infected or uninfected mammary glands elicited an influx of polymorphonuclear leukocytes exhibiting subsequent enhanced activity and increased the cure rate 20–30% in quarters infected with S. aureus.39
A novel method for decreasing the transmission of S. aureus within a herd is to selectively cease lactation in infected quarters of lactating cattle.40 The best method for permanently drying off a quarter is infusion of 120 mL of 5% povidone–iodine solution (0.5% iodine) after complete milk-out and administration of flunixin meglumine (1 mg/kg BW, intravenously). Therapeutic cessation of milk production in one quarter does not alter daily milk production but does decrease individual cow SCC and its contribution to the bulk tank milk SCC. The final outcome of selectively drying off infected quarters is a decrease in the rate of new intramammary infections in the herds and a lowering in the bulk tank milk SCC.
Early parenteral treatment of peracute cases with adequate does of antimicrobials such as trimethoprim– sulfonamide or penicillin is deemed necessary to improve the survival rate. When penicillin is used the initial intramuscular injection should be supported by an intravenous dose of crystalline penicillin, with subsequent intramuscular doses to maintain the highest possible blood level of the antimicrobial over a 4–6-day period; tamethicillin or penthemate hydriodide are preferred to achieve this. Intramammary infusions are of little value in such cases because of failure of the drugs to diffuse into the gland. The intravenous administration of large quantities of electrolyte solutions is also recommended. Hypertonic saline, as recommended for peracute coliform mastitis, has not yet been evaluated but may be indicated. Frequent massage of the udder with hot wet packs and milking out the affected gland is recommended. Oxytocin is used to promote milk-down but is relatively ineffective in severely inflamed glands. Surgical amputation of the teat may be indicated to promote drainage of the gland, but only in cows with necrotic teats.
It has become a common practice to leave chronic S. aureus cases until they are dried off before attempting to eliminate the infection. The material is infused into each gland after the last milking of the lactation and left in situ. The major benefits of dry cow therapy are the elimination of existing intramammary infections and prevention of new intramammary infections during the dry period. In addition, milk is not discarded and bacteriological cure rates are superior to those obtained during lactation.
The factors associated with a bacteriological cure after dry cow therapy of subclinical S. aureus mastitis have been examined.41 The probability of cure of an infected quarter decreased when:
• Another quarter was infected in the same cow
• The infection was in a hind quarter
• The percentage of samples that were positive for S. aureus was higher before drying off.
Cows with more than one infected quarter were 0.6 times less likely to be cured than cows with one infected quarter. The cure rate of quarters affected with S. aureus can be predicted using a formula which considers several cow factors and quarter factors.41 The prediction of the probability of cure in an 8-year-old cow with three quarters infected with the organism and a SCC of 2300000 is 36%. In a 3-year-old cow with one quarter infected and a SCC of 700000, the probability of cure is 92%. This information is often available at drying off and can be used to select cows that are unlikely to be cured to be removed from the herd by designating them as ‘do not breed’ and culling when it is economically opportune.
Most intramammary antimicrobial infusions are satisfactory for dry cow therapy provided they are combined with slow-release bases. Bacteriological cure rates vary between herds from 25–75% and average about 50%.42 The use of parenteral antimicrobials such as oxytetracycline along with an intramammary infusion of cephapirin did not improve the cure rate for S. aureus.42
Because of the relatively poor results obtained in the treatment of staphylococcal mastitis, any attempt at control must depend heavily on effective methods of preventing the transmission of infection from cow to cow. S. aureus is a contagious pathogen, the udder is the primary site of infection, and hygiene in the milking parlor is of major importance. To reduce the source of the organism, a program of early identification, culling, and segregation is important to control S. aureus mastitis in a dairy herd, although successful implementation of all three aspects is challenging. Satisfactory control of S. aureus mastitis has historically been difficult and unreliable; however, at the present time the quarter infection rate can be rapidly and profitably reduced from the average level of 30% to 10% or less.
The strategies and practices described under the control of bovine mastitis later in this chapter are highly successful for the control of S. aureus mastitis when applied and maintained rigorously. The control program includes:
• Hygienic washing and drying of udders before milking
• Regular milking-machine maintenance
• Teat dipping after milking. Teat dipping in 1% iodine or 0.5% chlorhexidine, either in 5–10% glycerine, is completely effective against S. aureus mastitis.43 The program helps to eliminate infected quarters and reduces the new infection rate by 50–65% compared to controls. The disinfection of hands or use of rubber gloves provides additional advantages
• Dry cow treatment on all cows
• Culling cows with chronic mastitis
• Milking infected cows last (very difficult to implement in free stall housing or pasture feeding).
An alternative but radical control strategy when all else has failed is to permanently dry off the infected quarter using iodine infusion.
Immunization against S. aureus mastitis has been widely researched for 100 years. Different vaccines based on cellular or soluble antigens with and without adjuvants have been given to dairy cows but protection against infection and clinical disease has been unsatisfactory when used in the field. Currently available vaccines are autogenous bacterins (made to order using isolates from clinical cases on the farm) or contain one or more S. aureus strains that are believed to provide good cross-protection. The goals of such vaccines are to decrease the severity of clinical signs and increase the cure rate, particularly when administered to heifers before they calve. Vaccination has also been used simultaneously with antimicrobial therapy during lactation or at dry-off in an attempt to augment the cows immune response, with mixed success.21 Development of an effective S. aureus vaccine remains one of the most important issues confronting control of infectious diseases in cattle.
1 Torgerson PR, et al. Vet Rec. 1992;130:54.
2 Roberson JR, et al. J Dairy Sci. 1994;77:3354.
3 Gillespie BE, et al. J Dairy Sci. 1999;82:1581.
4 Owens WE, et al. Am J Vet Res. 1998;59:1122.
5 Myllys V, et al. J Dairy Sci. 1994;77:446.
6 Fox LK, Cummings MS. J Dairy Res. 1996;63:369.
7 Nickerson SC, Boddie RL. J Dairy Sci. 1994;77:2526.
8 Enevoldsen C, et al. J Dairy Res. 1995;62:69.
9 Schukken YH, et al. J Dairy Sci. 1994;77:639.
10 Daley MJ, Hayes P. Cornell Vet. 1992;82:1.
11 Daley MJ, et al. Am J Vet Res. 1991;52:474.
12 Burton JL, Kehill MEJr. Am J Vet Res. 1995;56:997.
13 Bartlett PC, Miller GY. Prev Vet Med. 1993;17:33.
14 Almeida RA, et al. J Vet Med B. 1997;44:139.
15 Cifrian E, et al. Vet Microbiol. 1996;48:187.
16 Baselga R, et al. Vet Microbiol. 1994;39:195.
17 Aaerstrup FM, et al. Can J Vet Res. 1995;59:124.
18 Lam TJGM, et al. Am J Vet Res. 1996;57:39.
19 Aaerstrup FM, et al. Acta Vet Scand. 1995;38:243.
20 Fitzgerald JR, et al. Epidemiol Infect. 1997;119:261.
21 Luby CD, Middleton JR. Vet Rec. 2005;157:89.
22 Smith EM, et al. J Clin Microbiol. 2005;43:4737.
23 Aarestrup FM, et al. Acta Vet Scand. 1995;36:273.
24 Persson K, et al. J Vet Med B. 1995;42:435.
25 Hicks CR, et al. J Am Vet Med Assoc. 1994;204:253.
26 Buelow KL, et al. Prev Vet Med. 1996;25:343.
27 Buelow KL, et al. Prev Vet Med. 1996;26:1.
28 Lam TJGM, et al. J Am Vet Med Assoc. 1996;208:1705.
29 Ollis GW, et al. Can Vet J. 1995;36:619.
30 Middleton JR, et al. J Am Vet Med Assoc. 2004;224:419.
31 Matsushita T, et al. J Vet Diagn Invest. 1990;2:163.
32 Watts JL, Washburn PJ. J Clin Microbiol. 1991;29:59.
33 Wallace RL, et al. J Am Vet Med Assoc. 1998;213:394.
34 Owens WE, et al. J Dairy Sci. 1991;74:3376.
35 Wilson DJ, et al. J Dairy Sci. 1995;78:2083.
36 Lopes CAM, Moreno G. Res Vet Sci. 1991;51:339.
37 Wilson DJ, et al. J Dairy Sci. 1999;82:1664.
38 Owens WE, et al. J Dairy Sci. 1988;71:3143.
39 Sanchez MS, et al. J Dairy Sci. 1994;77:1251.
40 Middleton JR, Fox LK. J Dairy Sci. 2001;84:1976.
41 Sol J, et al. J Dairy Sci. 1994;77:75.
Etiology Streptococcus agalactiae is a major pathogen of the mammary gland and a common cause of contagious bovine mastitis
Epidemiology Major cause of mastitis in dairy herds without an effective mastitis control program. Prevalence of infection 10–50% of cows and 25% of quarters. In herds with effective control program prevalence less than 10% of cows. Can be eliminated from herd with treatment and control. Highly contagious obligate pathogen. Infection is transmitted at milking
Clinical findings Individual repeated episodes of subacute to acute mastitis are most common. Gland is swollen, warm, and milk is watery and contains clots. Gradual induration of udder if not treated
Clinical pathology Culture of individual cow milk samples or bulk tank milk samples. Latex agglutination test
Necropsy findings Not important
Diagnostic confirmation Latex agglutination test for specific identification of organism
Differential diagnosis Cannot differentiate clinically from other causes of acute and chronic mastitis. Must culture milk
Treatment Mastitis associated with S. agalactiae in lactating cows is sensitive to intramammary therapy with wide variety of antimicrobial agents resulting in high rate of clinical and bacteriological cures. Blitz therapy (simultaneous treatment of all positive cows in a herd) commonly used to reduce prevalence of infection in herd
Control Eradication is possible. Identify and treat infected quarters, cull incurable cows. Premilking teat and udder sanitation, postmilking teat dipping, and dry cow therapy
Streptococcus agalactiae. Infections with environmental streptococci are described in the next section.
S. agalactiae was the major cause of mastitis before the antimicrobial era and is still a significant cause of chronic mastitis where control procedures for contagious mastitis are not used.1 Herd prevalence rates of infection range from 11–47%. Typically, in a herd infected with the pathogen, the prevalence of infection could be as high as 50% of cows, but more recent surveys indicate much lower within-herd prevalences, ranging from 8–10%.1 Where good hygienic measures and the efficient treatment of clinical cases are in general use, the prevalence of infection within a herd will be less than 10% of cows. Following the use of antimicrobial agents, S. agalactiae was superseded by Staphylococcus aureus as the major cause of bovine mastitis. In herds with a high bulk tank milk SCC, the probability is high that S. agalactiae infection is the most prevalent pathogen.
S. agalactiae is a highly contagious obligate parasite of the bovine mammary gland. The main source of infection is the udder of infected cows although, when hygiene is poor, contamination of the environment may provide an additional source. The teats and skin of cattle, milkers’ hands, floors, utensils and clothes are often heavily contaminated. Sores on teats are the commonest sites outside the udder for persistence of the organism. The infection may persist for up to 3 weeks on hair and skin and on manure and bricks. The importance of environmental contamination as a source of infection is given due recognition in the general disinfection technique of eradication.
Transmission from animal to animal occurs most commonly by the medium of milking machine liners, hands, udder cloths and possibly bedding.
The streak canal is the portal of entry, although there is doubt as to how invasion into the teat canal and then gland occurs. Suction into the teat during milking or immediately afterwards does occur, but growth of the bacteria into the canal between milkings also appears to be an important method of entry. It is difficult to explain why heifers that have never been milked may be found to be infected with S. agalactiae, although sucking between calves after ingestion of infected milk or contact with infected inanimate materials may be sources of infection.
There is no particular breed susceptibility but infection does become established more readily in older cows and in the early part of each lactation. Poor hygiene, incompetent milking personnel and machinery that is faulty or maladjusted are important risk factors. The most important risk factors are the failure to use postmilking teat dip and the selective or non-use of dry cow therapy. The use of a common wash rag or sponge is also a risk factor. Inadequate treatment of clinical cases of mastitis is also a frequent risk factor in infected herds.
S. agalactiae has the ability to adhere to the mammary gland tissue, and the specific microenvironment of the udder is necessary for growth of the organism. The virulence of various strains of the organism is related to differences in their ability to adhere to the mammary epithelium. Bacterial ribotyping has been used to characterize strains of the organism to determine their geographical distribution.2 The physical characteristics of the teat canal may influence the susceptibility to streptococcal infection.3 The mechanisms used by S. agalactiae to penetrate the teat canal are influenced more by the diameter of the teat canal lumen, as reflected by the peak flow rate, than by teat canal length.
The disease is of major economic importance in milk production. In individual cows, the loss of production associated with S. agalactiae mastitis is about 25% during the infected lactation, and in affected herds the loss may be of the order of 10–15% of the potential production. Reduction of the productive life represents an average loss of one lactation per cow in an affected herd. Deaths due to S. agalactiae infection rarely if ever occur and complete loss of productivity of a quarter is uncommon, the losses being incurred in the less dramatic but no less important fashion of decreased production per cow.
When the primary barrier of the streak canal is passed, if bacteria are not flushed out by the physical act of milking they proliferate and invasion of the udder tissue follows. There is considerable variation between cows in the developments that occur at each of the three stages of invasion, infection and inflammation. The reasons for this variation are not clear but resistance appears to depend largely on the integrity of the lining of the teat canal. After the introduction of infection into the teat, the invasion, if it occurs, takes 1–4 days and the appearance of inflammation 3–5 days. Again there is much variation between cows in the response to tissue invasion, and a balance may be set up between the virulence of the organism and undefined defense mechanisms of the host so that very little clinically detectable inflammation may develop despite the persistence of a permanent bacterial flora.
The development of mastitis associated with S. agalactiae is essentially a process of invasion and inflammation of lobules of mammary tissue in a series of crises, particularly during the first month after infection, each crisis developing in the same general pattern. Initially there is a rapid multiplication of the organism in the lactiferous ducts, followed by passage of the bacteria through the duct walls into lymphatic vessels and to the supramammary lymph nodes, and an outpouring of neutrophils into the milk ducts. At this stage of initial tissue invasion, a shortlived systemic reaction occurs and the milk yield falls sharply as a result of inhibition and stasis of secretion caused by damage to acinar and ductal epithelium. Fibrosis of the interalveolar tissue and involution of acini result even though the tissue invasion is quickly cleared. Subsequently, similar crises develop and more lobules are affected in the same way, resulting in a stepwise loss of secretory function with increasing fibrosis of the quarter and eventual atrophy.
The clinicopathological findings vary with the stage of development of the disease. Bacterial counts in the milk are high in the early stages but fall when the SCC rises at the same time as swelling of the quarter becomes apparent. In some cases bacteria are not detectable culturally at this acute stage. The SCC rises by 10–100 times normal during the first 2 days after infection and returns to normal over the next 10 days. The febrile reaction is often sufficiently mild and shortlived to escape notice. When the inflammatory changes in the epithelial lining of the acini and ducts begin to subside, the shedding of the lining results in the clinical appearance of clots in the milk. Thus the major damage has already been done when clots are first observed. At the stage of acute swelling, it is the combination of inflamed interalveolar tissue and retained secretion in distended alveoli that causes the swelling. Removal of the retained secretion at this stage may considerably reduce the swelling and permit better diffusion of drugs infused into the quarter. Inflammatory reactions also occur in the teat wall of affected quarters.
The variations in resistance between cows and the increased susceptibility with advancing age are unexplained. Hormonal changes and hypersensitivity of mammary tissue to streptococcal protein have both been advanced as possible causes of the latter. Local immunity of mammary tissue after an attack probably does not occur but there is some evidence to suggest that a low degree of general immunity may develop. The rapid disappearance of the infection in a small proportion of cows in contrast to the recurrent crises that are the normal pattern of development suggests that immunity does develop in some animals. The antibodies are hyaluronidase inhibitors and are markedly specific for specific strains of the organism. A nonspecific rise in other antibodies may occur simultaneously and this is thought to account for the field observations that coincident streptococcal and staphylococcal infections are unusual and that the elimination of one infection may lead to an increased incidence of the other.
In the experimentally produced disease, there is initially a sudden episode of acute mastitis, accompanied by a transient fever, followed at intervals by similar attacks, which are usually less severe. In natural cases fever, lasting for a day or two, is occasionally observed with the initial attack but the inflammation of the gland persists and the subsequent crises are usually of a relatively mild nature. These degrees of severity may be classified as abnormal cow when the animal is febrile and off its feed, abnormal gland when the inflammation of the gland is severe but there is no marked systemic reaction, and abnormal milk when the gland is not greatly swollen, pain and heat are absent and the presence of clots in watery foremilk may be the only apparent abnormality. Induration is most readily palpable at the udder cistern and in the lower part of the udder, and varies in degree with the stage of development of the disease.
The milk yield of affected glands is markedly reduced during each crisis but, with proper treatment administered early, the yield may return to almost normal. Even without treatment the appearance of the milk soon becomes normal but the yield is significantly reduced and subsequent crises are likely to reduce it further.
The CAMP test, which has served as the universally used means of identifying S. agalactiae for many years, has been displaced by a commercial latex agglutination test, which contains specific reagents necessary for the identification of S. agalactiae and is suitable for general laboratory use.1 When used on isolates of samples from bulk tank milk, the sensitivity and specificity are 97.6% and 98.2%, respectively. An ELISA test correlates well with the bulk tank milk SCC and provides a suitable alternative.1
The critical judgment to be made is deciding when the quarter infection rate is so high that control or eradication measures are necessary. A decision can be made on the basis of the bulk tank milk SCC as an indicator of the prevalence of mastitis on a quarter basis, and on culture of the bulk tank milk sample to indicate that S. agalactiae is the important pathogen, but this approach is too inaccurate to be recommended. There seems to be no alternative to carrying out bacteriological culture and determining SCC on milk samples from individual cows or quarters. Milk samples collected for bacteriological examination for the presence of S. agalactiae can be stored in the frozen state. The number of samples that will be culturally positive when the stored frozen samples are thawed will either be unchanged or enhanced up to 200%; the latter response is attributing to fracturing of cellular debris containing S. agalactiae.
The presence of the organism in bulk tank milk is due to shedding of bacteria from infected quarters, with cyclic shedding being typical. The specificity of culture from bulk tank milk is very high; the sensitivity is much lower but can be increased by using selective media.
The total bacterial count in bulk tank milk can be markedly increased due to the presence of S. agalactiae mastitis in the herd. Samples of bulk tank milk from infected herds commonly contain bacterial counts in the range of 20000–100000cfu/mL, because a cow in the early stages of infection can shed up to 100000000 bacteria/mL. The standard plate count can drop from 100000 to 2000 cfu/mL after implementation of a modified blitz therapy and control program to control S. agalactiae.
The gross and microscopic pathology of mastitis associated with S. agalactiae are not of importance in the diagnosis of the disease.
S. agalactiae is very sensitive to intramammary therapy using a wide variety of commercially available intramammary infusion preparations. Systemic therapy is also effective but offers no advantages over the intramammary route. Clinical cases should be treated whenever they occur because of the need to prevent transmission to uninfected quarters and cows. Subclinical cases identified at any stage of lactation should be treated immediately because of the excellent response to treatment. Treatment of S. agalactiae mastitis with intramammary infusions will result in a high percentage of infections being eliminated economically and with few residual concerns, provided the milk withholding times are observed.
Infections at all stages of lactation have 90–100% cure rates with penicillin, erythromycin, cloxacillin and cephalosporins. Gentamicin, neomycin, nitrofurazone and polymyxin B have poor activity. Procaine penicillin G is universally used as a mammary infusion at a dose rate of 100000 units. Higher dose rates have the disadvantage of increasing penicillin residues in the milk. A moderate increase in efficiency is obtained by using procaine penicillin rather than the crystalline product, and by using 100000 units of penicillin in a long-acting base the cure rate (96%) is significantly better than with quick-acting preparations (83%).
To provide a broader spectrum of antimicrobial efficiency penicillin is often combined with other drugs that are more effective against Gram-negative organisms. A mixture of penicillin (100000 units) and novobiocin (150 mg) provides a cure rate ranging from 89–98%.4 It is necessary to maintain adequate milk levels for 72 hours: three infusions at intervals of 24 hours are recommended, but dosing with two infusions 72 hours apart, or one infusion of 100000 units, in a base containing mineral oil and aluminum monostearate, gives similar results. As a general rule clinical cases should be treated with three infusions, and subclinical cases, particularly those detected by routine examination in a control program, with one infusion. Recovery, both clinically and bacteriologically, should be achieved in at least 90% of quarters if treatment has been efficient. Intramuscular administration of ceftiofur is not efficacious as a treatment to eliminate the organism, compared to intramammary infusion of penicillin (100000 units) and novobiocin (150 mg) daily for two treatments.4
Other antimicrobial agents used in the treatment of S. agalactiae infections include the tetracyclines and cephalothin, which are as effective as penicillin and have the added advantage of a wider antibacterial spectrum, an obvious advantage when the type of infection is unknown. Neomycin is inferior to penicillin in the treatment of S. agalactiae mastitis, while tylosin and erythromycin appear to have equal efficacy. A single treatment with 300 mg of erythromycin is recommended as curing 100% of quarters infected with S. agalactiae. Lincomycin (200 mg) combined with neomycin (286 mg) and administered twice at 12-hour intervals also has good efficacy. In a study of 1927 cases of subclinical S. agalactiae mastitis in New York, all commercially available intramammary infusions were more effective than untreated controls (27% bacteriological cure), with the following bacteriological cure rates: amoxicillin (86%), erythromycin (81%), cloxacillin (77%), cephapirin (66%), penicillin (63%), hetacillin (62%), pirlimycin (44%).5
In dry cows, one infusion is sufficient, milk levels of penicillin remaining high for 72 hours. Cloxacillin eliminated the organism from 98% and 100% of infected cows in two different studies.1
The prevalence of subclinical mastitis due to S. agalactiae can be reduced more rapidly by treatment of infected cows during lactation than by dry cow therapy and postmilking teat dipping. S. agalactiae is one of the few pathogens causing subclinical mastitis that can be treated economically during lactation, and can be eliminated from herds with blitz antimicrobial therapy followed by good sanitation procedures. All cows are sampled and those that are positive are treated simultaneously with penicillin and novobiocin. Cows not responsive to the first treatment are identified and retreated or culled. Failure to institute sanitation procedures for the control of the pathogen may result in subsequent outbreaks of mastitis.6
If blitz therapy of all infected cows is not possible because of the short-term effect of lost milk production on income, a modified treatment protocol is recommended. The herd is divided into two groups, based on a composite milk SCC of 500000. Those cows in the high category are treated with 300 mg of erythromycin, intramammarily. When lactating cow numbers reach their lowest point, all animals are treated with the same product. At drying off, cows are treated with 500 mg cloxacillin and 250 mg ampicillin.
Eradication on a herd basis of mastitis associated with S. agalactiae is an accepted procedure and has been undertaken on an area scale in some countries. The control measures as outlined later in this chapter are designed especially for this disease and should be adopted in detail. If suitable hygienic barriers against infection can be introduced and if the infection can be eliminated from individual quarters by treatment, the disease is eradicable fairly simply and economically.
The control program consists of:
The control program is particularly applicable in herds where an unacceptable level of clinical cases is backed by a high incidence of subclinical infections. Premilking teat and udder sanitation, postmilking teat dipping, and dry cow therapy are vital aspects of the control program.
Vaccination against S. agalactiae has been attempted and elicits systemic hyperimmunity but no apparent intramammary resistance. Development of an effective vaccine will be difficult because of the multiplicity of strains involved and the known variability between animals in their reaction to intramammary infection.
As with any eradication program a high degree of vigilance is required to maintain a ‘clean’ status. This is particularly so with mastitis due to S. agalactiae. Breakdowns are usually due to the introduction of infected animals, even heifers that have not yet calved, or the employment of milkers who carry infection with them. Most dairy farms in the USA are in an ongoing process of herd expansion or replacement acquisition by the addition of purchased animals. Introduction of contagious mastitis associated with S. agalactiae, S. aureus and M. bovis is a common result. It has been recommended that herd additions should be screened for these important pathogens;7 however, currently available screening tests do not have perfect sensitivity.
1 Keefe GP. Can Vet J. 1997;38:429.
2 Rivas AL, et al. Am J Vet Res. 1997;58:482.
3 Lacy-Hubert SJ, Hillerton JE. J Dairy Res. 1995;62:395.
4 Erskine RJ, et al. J Am Vet Med Assoc. 1996;208:258.
5 Wilson DJ, et al. J Dairy Sci. 1999;82:1664.
6 Boyer PJ. Vet Rec. 1997;141:55. 84, 108
7 Wilson DJ, Gonzalez RN. In: Proceedings of 36th Annual Meeting of National Mastitis Council, 1997:127.
A number of species of Mycoplasma, especially M. bovis and occasionally Mycoplasma species group 7,1 Mycoplasma F-38, Mycoplasma arginini, Mycoplasma bovirhinis, Mycoplasma canadensis, Mycoplasma bovigenitalium, Mycoplasma alkalescens,2 Mycoplasma capricolium,3 Mycoplasma californicum4,5 and Mycoplasma dispar,6 have been isolated from clinical cases. Other mycoplasmas, not usually associated with the development of mastitis, also cause the disease when injected into the udder. There is also evidence of mastitis associated with Ureaplasma spp.7 A striking characteristic of the mycoplasmas is that they seem to be able to survive in the presence of large numbers of leukocytes in the milk. Antibodies to the bacteria have not been detectable in sera or whey from animals infected with some strains, but complement-fixing antibodies are present in the sera of animals recovered from infection with other strains.
Etiology Mycoplasma bovis, other Mycoplasma spp.
Epidemiology A highly contagious mastitis causing outbreaks of clinical mastitis. Most common in large herds with recent introductions. Transmitted within herds by bulk mastitis treatments and poor milking hygiene. Cows of all ages and any stage of lactation but those in early lactation most severely affected
Clinical findings Sudden onset of clinical mastitis in many cows, usually all four quarters, marked drop in milk production and may stop lactating, swelling of the udder and gross abnormality of the milk without obvious signs of systemic illness, eventually udders atrophy and do not return to production. Can cause clinical, subclinical and chronic intramammary infections. Calves suckling milk from infected cows may develop otitis media/interna
Clinical pathology Special culture and staining of milk techniques
Necropsy findings Purulent interstitial mastitis
Diagnostic confirmation Identification of pathogen in milk
Differential diagnosis Epidemiology and clinical findings are characteristic of mycoplasma mastitis. May resemble other causes of chronic mastitis unresponsive to treatment
Treatment Not responsive to commonly used mastitis treatments protocols. Identify and cull affected cows for slaughter
Control Prevent entry of infected cows into herd. Eradicate infection by culling affected cows
Acholeplasma laidlawii is not a mastitis pathogen, but it has been observed that a high proportion of bulk tanks will give positive cultural tests for it, especially during wet, rainy weather. This increase is accompanied by an increase of clinical mycoplasmal mastitis due to pathogenic mycoplasma. A. laidlawii is considered to be a milk contaminant in these circumstances.
The group of diseases, including mastitis, that are associated with Mycoplasma spp. in sheep and goats are dealt with separately.
The disease has been recorded since the mid 1960s in the USA, Canada, UK, and Israel and has been observed in Australia. The quarter infection rate in infected herds varies widely.
The epidemiology of the disease has been incompletely characterized.8 Mycoplasma mastitis occurs most commonly in large herds and in herds where milking hygiene is poor and when cows are brought in from other farms or from public saleyards. Mycoplasma mastitis usually breaks out subsequently after a delay of weeks or even months. The delay in development of an outbreak may be related to the long-term persistence of the organism (more than 12 months) in some quarters, and some cows become shedders of the organism without ever exhibiting signs of severe clinical mastitis. M. bovis was isolated from milk samples of 5–12% of cows during two lactations and two dry periods.9
M. bovis is capable of colonizing and surviving in the upper respiratory tract and the vagina, and extramammary colonization explains many of its epidemiological paradoxes. An interesting epidemiological observation is the detection of mycoplasmas and infectious bovine rhinotracheitis virus in affected udders at the same time. The virus could be the much sought-after unknown factor in the etiology of the disease. Outbreaks of mastitis are recorded concurrently with outbreaks of vaginitis and otitis media/interna vestibulitis.10
Entry of the disease to a herd is usually by the purchase of animals and their introduction without quarantine. Transmission within a herd is most commonly at milking via machine milking or the hands of milkers. Transmission can also be through the use of bulk mastitis treatments administered through a common syringe and cannula.11 Although the disease occurs first in the inoculated quarter there is usually rapid spread to all other quarters.
Hematogenous spread of M. bovis has been demonstrated.12,13 Colonization of body sites other than the mammary gland is common, and M. bovis isolates from the respiratory and urogenital systems are frequently the same M. bovis subtypes that cause mastitis.13
Mycoplasma spp. group 7 has also been isolated from cases of pneumonia and polyarthritis in calves fed milk from cows with mycoplasmal mastitis.
Cows of all ages and at any stage of lactation are affected, cows that have recently calved showing the most severe signs and dry cows the least. There are several recorded outbreaks in dairy herds in dry cows.14 one of them immediately after mammary infusions of dry period treatment that affected all quarters of all cows.
Experimental production of the disease15 with M. bovis causes severe loss of milk production, a positive CMT reaction and clots in the milk.16 Experimental infection produces little tissue necrosis but Mycoplasma are detectable in many tissues, including blood, vagina, and fetus, indicating that hematogenous spread has occurred. It is also apparent that spread of infection between quarters in one cow can be hematogenous. There are no significant pathological differences between mastitis produced by M. bovigenitalium and M. bovis; however, M. bovis remains the most common cause of mycoplasmal mastitis in dairy cattle.
This is a purulent interstitial mastitis. Although infection probably occurs via the streak canal, the rapid spread of the disease to other quarters of the udder and occasionally to joints suggests indicates that hematogenous spread may occur. The presence of the infection in heifers milked for the first time also suggests that systemic invasion may be followed by localization in the udder.
In lactating cows, there is a sudden onset of swelling of the udder, a sharp drop in milk production and grossly abnormal secretion in one or more quarters. In most cases all four quarters are affected and a high-producing cow may fall in yield to almost nil between one milking and the next. Dry cows show little swelling of the udder. Although there is no overt evidence of systemic illness, and febrile reactions are not observed in most field cases in lactating cows, those that have recently calved show most obvious swelling of the udder and may be off their feed and have a mild fever. However, cows infected experimentally show fever up to 41°C (105.5°F) on the third or fourth day after inoculation, at the same time as the udder changes appear. The temperature returns to normal in 24–96 hours. In some cases the supramammary lymph nodes are greatly enlarged. The classic clinical presentation is severe clinical mastitis in multiple quarters of multiple cows with minimal systemic signs of disease. A few cows, with or without mastitis, develop arthritis in the knees and fetlocks. The affected joints are swollen, with the swelling extending up and down the leg. Lameness may be so severe that the foot is not put to the ground. Mycoplasma may be present in the joint.
The secretion from affected quarters is deceptive in the early stages in that it appears fairly normal at collection; on standing, however, a deposit, which may be in the form of fine, sandy material, flakes or floccules, settles out leaving a turbid whey-like supernatant. Subsequently the secretion becomes scanty and resembles colostrum or soft cheese curd in thin serum. The secretion may be tinged pink with blood or show a gray or brown discoloration. Within a few days the secretion is frankly purulent or curdy but there is an absence of large, firm clots. This abnormal secretion persists for weeks or even months.
Affected quarters are grossly swollen. Response to treatment is very poor and the swollen udders become grossly atrophied. In infection with one strain of the Mycoplasma, many cows do not subsequently come back into production although some may produce moderately well at the next lactation. With other strains there is clinical recovery in 1–4 weeks without apparent residual damage to the quarter.
Mycoplasmal mastitis due to M. bovigenitalium may be very mild and disappear from the herd spontaneously and without causing loss of milk production.17
The causative organism can be cultured without great difficulty by a laboratory skilled in working with Mycoplasma. Samples for culture should be freshly collected and transported at 4°C, and concurrent infection with other bacteria is common. Diagnosis at the herd level can be made by culturing bulk tank milk or milk from cows with clinical mastitis or increased SCC. However, the sensitivity of bulk tank milk culturing is poor (33–59%).18,19 A marked leukopenia, with counts as low as 1800–2500 cells/μL, is present when clinical signs appear and persists for up to 2 weeks. Somatic cell counts in the milk are very high, usually over 20000000 cells/mL. In the acute stages the organisms may be able to be visualized by the examination of a milk film stained with Giemsa or Wright– Leishman stain. Species identification of Mycoplasma isolates is most commonly done using immunofluorescence and homologous fluorescein-conjugated antibody or an indirect immunoperoxidase test (immunohistochemistry). Speciation of the causative Mycoplasma species is recommended.
Grossly, diffuse fibrosis and granulomatous lesions containing pus are present in the mammary tissue. The lining of the milk ducts and the teat sinus is thick and roughened. On histological examination the granulomatous nature of the lesions is evident. Metastatic pulmonary lesions have been found in a few long-standing cases.
A presumptive diagnosis can be made based on the clinical findings, but laboratory confirmation by culture of the organism is desirable. The facts that the organism does not grow on standard media and that other pathogenic bacteria are commonly present often lead to errors in the laboratory diagnosis unless attention is drawn to the characteristic field findings.
The majority of M. bovis strains isolated from cattle are susceptible in vitro to fluoroquinolones, florfenicol and tiamulin.20,21 Approximately half of the isolates are susceptible to spectinomycin, tylosin and oxytetracycline, and very few isolates are susceptible in vitro to gentamicin, tilmicosin, ceftiofur, ampicillin, or erythromycin.20,21 The clinical relevance of these in vitro susceptibility data to treating mycoplasmal mastitis remains questionable.
Cows diagnosed with mycoplasmal mastitis should be considered to be infected for life. None of the commonly used antimicrobial agents appear to be effective and oil–water emulsions used as intramammary infusions appear to increase the severity of the disease. Parenteral treatment with oxytetracycline (5 g intravenously, daily for 3 days) has been shown to cause only temporary improvement. A mixture of tylosin 500 mg and tetracycline 450 mg used as an infusion cured some quarters.22 Unless treatment is administered very early in the course of the disease, the tissue damage has already been done.
Prevention of introduction of the disease into a herd appears to depend upon avoidance of introductions, or isolating introduced cows until they can be checked for mastitis. A popular biosecurity recommendation is to culture the milk of all replacement cows for M. bovis, but the sensitivity and specificity of milk culture in cows with subclinical infections appears to be low. The disease spreads rapidly in a herd and affected animals should be culled immediately or placed in strict isolation until sale. Eradication of the disease can be achieved by culling infected cows identified by culture of milk and nasal swabs, especially at drying off and calving. When eradication is completed the bulk tank milk SCC is the best single monitoring device to guard against reinfection. An alternative program recommended for large herds is the creation of an infected subherd that is milked last. There appears to be merit in the frequent culturing of bulk milk samples as a surveillance strategy for problem herds and areas. Frequent culturing overcomes the poor sensitivity of bulk tank milk culturing. Cows with infected quarters are segregated into the subherd and cows developing clinical illness or decreased milk yield are culled.23
Intramammary infusions must be carried out with great attention to hygiene and preferably with individual tubes rather than multidose syringes. Most commercial teat dips are effective in control. Use of disposable latex gloves with disinfection of the gloved hands between cows may minimize transmission at milking.
Vaccination is a possible development but is unlikely to be a satisfactory control measure because the observed resistance of a quarter to infection after a natural clinical episode is less than 1 year. A M. bovis bacterin is commercially available in the USA that contains multiple strains of M. bovis. Autogenous bacterins have also been made for specific herds; however, no vaccine has proven efficacy for preventing, decreasing the incidence of, or decreasing the severity of clinical signs of mycoplasmal bovine mastitis.24
Mycoplasma are sensitive to drying and osmotic changes, but more resistant than bacteria to the effects of freezing or thawing. Amputating the teats of affected quarters may result in heavy contami-nation of the environment and is not recommended. Because M. bovis can cause respiratory disease, otitis media/interna and arthritis in calves, all colostrum and waste milk fed to calves should be pasteurized.
1 Alexander PG, et al. Aust Vet J. 1985;62:135.
2 Jackson G, et al. Vet Rec. 1981;108:31.
3 Taoudi A, Kirchhoff H. Vet Rec. 1986;119:247.
4 Pfutzner H, et al. Arch Exp Vet Med. 1986;40:56.
5 Mackie DP, et al. Vet Rec. 1982;110:578.
6 Hodges RT, et al. N Z Vet J. 1983;31:60.
7 Jurmanova K, et al. Arch Exp Vet Med. 1986;40:67.
8 Feenstra A, et al. J Vet Med B. 1991;38:195.
9 Gonzalez RN, Sears PM. Proc Annu Conv Am Assoc Bovine Pract. 1994;26:184.
10 Pfutzner H, et al. Monatsh Vet Med. 1986;41:382.
11 Gonzalez RN, et al. Cornell Vet. 1992;82:29.
12 Pfützner H, Schimmel D. J Vet Med A. 1985;32:265.
13 Biddle MK, et al. J Am Vet Med Assoc. 2005;227:455.
14 Mackie DP, et al. Vet Rec. 1986;119:350.
15 Ball HJ, et al. Ir Vet J. 1994;47:45.
16 Boothby JT, et al. Can J Vet Res. 1986;50:200.
17 Jackson G, Boughton E. Vet Rec. 1991;129:444.
18 González RN, et al. J Am Vet Med Assoc. 1986;189:442.
19 González RN, et al. J Am Vet Med Assoc. 1988;193:323.
20 Rosenbusch RF, et al. J Vet Diagn Invest. 2005;17:436.
21 Thomas A, et al. Vet Rec. 2003;153:428.
22 Ball HJ, Campbell AN. Vet Rec. 1989;125:377.
23 Brown MB, et al. J Am Vet Med Assoc. 1990;196:1097.
24 González RN, Wilson DJ. Vet Clin North Am Large Anim Pract. 2003;19:199.
Corynebacterium bovis is a common and very contagious pathogen that is most commonly associated with subclinical infection. However, C. bovis has been cultured from dairy cattle with clinical mastitis in 1.7% of cases1 and, in a herd that had controlled contagious mastitis pathogens, C. bovis was the only pathogen isolated in 22% of clinical mastitis episodes.2 There is considerable debate about the significance of C. bovis infections for mammary gland health and cow productivity. For this reason, C. bovis is classified as a minor pathogen.
The main reservoir of infection appears to be infected glands and teat ducts, and C. bovis spreads rapidly from cow to cow in the absence of adequate teat dipping. C. bovis is extremely contagious and the duration of intramammary infection is long (many months). The prevalence of C. bovis is typically low in herds using an effective germicidal teat dip, good milking hygiene and dry cow therapy.
In vivo and in vitro studies have demonstrated that the bacteria has a predilection for the streak canal, and this predilection has been associated with a requirement for lipids (possibly in the keratin plug) for growth.3 It is possible that C. bovis infection in the streak canal may compete with ascending bacterial infections for nutrients and thereby decrease the new intramammary infection rate. Alternatively, the mild increase in SCC associated with C. bovis infection might increase the ability of the quarter to respond to a new intramammary infection.
Intramammary infection with a minor pathogen induces a higher than normal SCC and thereby increases the resistance of the colonized quarter to invasion by a major pathogen. In particular, the lowest rate of intramammary infection with major pathogens is observed in quarters infected with C. bovis.4
An intramammary infection with C. bovis is infrequently associated with clinical disease but usually causes a moderate increase in the SCC and a small increase in the CMT score. Milk production losses are usually not detectable, and the mastitis is typically a thicker than normal milk (abnormal milk); occasional cases also have a large firm gland (abnormal gland).2 There are clear herd to herd differences in the apparent clinical pathogenicity of C. bovis, suggesting that strains of different virulence are present.
C. bovis is very susceptible to penicillin, ampicillin, amoxicillin, cephapirin, and erythromycin, and most other commercially available intramammary infusions. There is no need for parenteral treatment. The duration of infection is prolonged (months) in animals not treated with antimicrobial agents.
Mastitis of cattle associated with teat skin opportunistic pathogens
Because of the intense investigation of coagulase positive staphylococcal mastitis (S. aureus), coagulase-negative staphylococcal intramammary infections have come under closer scrutiny and are now among the most common bacteria found in milk, especially in herds in which the major pathogens have been adequately controlled. There is considerable debate about the significance of these pathogens for the mammary gland and for cow productivity. For this reason, these pathogens are classified as minor pathogens.
Coagulase-negative staphylococci are common but minor contagious pathogens that include Staphylococcus epidermidis, S. hyicus, S. chromogenes, S. simulans, and Staphylococcus warneri that are normal teat skin flora, and Staphylococcus xylosus and Staphylococcus sciuri that come from an uncertain site.
Coagulase-negative staphylococci are teat skin opportunistic pathogens and cause mastitis by ascending infection via the streak canal. Coagulase-negative staphylococci appear to have a protective effect against colonization of the teat duct and teat skin by S. aureus and other major pathogens,1 with the exception of E. coli and the environmental streptococci.
Studies in the US found that 20–70% of heifer quarters are infected before parturition with coagulase-negative staphylococci,2,3 but these infections are usually eliminated spontaneously or with antimicrobial therapy during early lactation. A survey of the prevalence and duration of intramammary infection in heifers in Denmark in the peripartum period found S. chromogenes in 15% of all quarters before parturition, but this decreased to 1% of all quarters shortly after parturition.2 In Finland, coagulase-negative staphylococci are the most commonly isolated bacteria from milk samples of heifers with mastitis.4 Infections with S. simulans and S. epidermidis occurred in 1–3% of quarters both before and after parturition.2 Infection with S. simulans persisted in the same quarter for several weeks, but intramammary infections with S. epidermidis were transient.
Coagulase-positive S. hyicus and Staphylococcus intermedius have been isolated from some dairy herds and can cause chronic, low-grade intramammary infection and be confused with S. aureus.5 The prevalence of infection with S. hyicus was 0.6% of all cows and 2% of heifers at parturition; the prevalence of infection of S. intermedius was less than 0.1% of cows.
Coagulase-negative staphylococci are usually associated with mild clinical disease (abnormal secretion only, occasionally abnormal gland) and are commonly isolated from mild clinical cases of mastitis and subclinical infections. For example, Staphylococcus spp. have been cultured from dairy cattle with clinical mastitis in 29% of cases,6 and subclinical infections usually induce a moderate increase in SCC.
Intramammary infections by minor pathogens such as coagulase-negative staphylococci result in a higher than normal SCC and thereby increase the resistance of the colonized quarter to invasion by a major pathogen.7 Although these bacteria are capable of causing microscopic lesions, they are not nearly as pathogenic as S. aureus,8 and necropsy reports are lacking.
Spontaneous cure is common. Coagulase-negative staphylococci, including S. chromogenes, S. hyicus and others, are very susceptible to penicillin, ampicillin, amoxicillin, clavulanic acid, cephapirin, erythromycin, gentamicin, potentiated sulfonamides and tetracyclines. In a study of 139 cases of subclinical coagulase-negative staphylococcal mastitis in New York, the bacteriological cure rates of commercially available intramammary infusions were similar to that of untreated controls (72% bacteriological cure), with the following bacteriological cure rates: cephapirin (89%), amoxicillin (87%), cloxacillin (76%) and penicillin (68%).9
The use of a combination of novobiocin and penicillin, and cloxacillin as dry cow therapy for coagulase-negative staphylococci gave cure rates of over 90%.10
1 Matthews KR, et al. J Dairy Sci. 1991;74:1855.
2 Aarestrup FM, Jensen NE. J Dairy Sci. 1997;80:307.
3 Erskine RJ, et al. J Dairy Sci. 1994;77:3347.
4 Myllys V. J Dairy Res. 1995;62:51.
5 Roberson JR, et al. Am J Vet Res. 1996;57:54.
6 Sargeant J, et al. Can Vet J. 1998;39:33.
7 Fox LK, et al. J Am Vet Med Assoc. 1996;209:1143.
8 Jarp J. Vet Microbiol. 1991;27:151.
Mastitis of cattle associated with common environmental pathogens
Environmental mastitis is associated with bacteria that are transferred from the environment to the cow rather than from other infected quarters. E. coli, Klebsiella spp. and environmental streptococci are the major pathogens causing environmental mastitis.
Many different serotypes of E. coli, numerous capsular types of Klebsiella spp. (most commonly K. pneumoniae) and Enterobacter aerogenes are responsible for coliform mastitis in cattle. E. coli isolated from the milk of cows with acute mastitis cannot be distinguished as a specific pathogenic group on the basis of biochemical and serological test reactions. The incidence of antimicrobial resistance is also low in these isolates because they are opportunists originating from the alimentary tract, from which antimicrobial resistant E. coli are rarely found in adults. Other Gram-negative bacteria which are not categorized as coliforms but can cause mastitis include Serratia, Pseudomonas, and Proteus spp.
Etiology Many different serotypes of Escherichia coli, numerous capsular types of Klebsiella spp. and Enterobacter aerogenes. These are commonly called coliform bacteria; other Gram-negative bacteria (such as Pseudomonas aeruginosa) can cause environmental mastitis but are not categorized as coliform bacteria.
Epidemiology Dairy cattle housed in total confinement or drylot; uncommon in pastured cattle. Most important mastitis problem in well managed, low-SCC herds. Quarter infection rate low at 2–4%. Incidence highest in early lactation. 80–90% of coliform infections result in clinical mastitis; 8–10% are peracute. Causes clinical mastitis rather than subclinical mastitis. Source of infection is environment between milkings, during dry period and prepartum in heifers. Isolates of E. coli are opportunists. Sawdust and shavings bedding contaminated with E. coli and Klebsiella spp. (particularly K. pneumoniae) major source of bacteria; much worse when wet (rainfall or high humidity). Coliform intramammary infection highest during 2 weeks following drying off and in 2 weeks prior to calving. Animal risk factors include:
Outbreaks of coliform mastitis do occur, commonly associated with major change in management of the environment (introduction of sawdust for bedding may result in outbreaks of Klebsiella mastitis).
Clinical findings Acute – swelling of gland, watery milk with small flakes, mild systemic response, recovery in few days. Peracute – sudden onset of severe toxemia, fever, tachycardia, impending shock; cow may be recumbent. Quarter may or may not be swollen and warm, secretions thin and serous and contain very small flakes. May die in few days.
Clinical pathology Culture milk. Somatic cell count. Marked leukopenia, neutropenia and degenerative left shift. Bacteremia may occur, particularly in severely affected cattle.
Necropsy findings Edema, hyperemia, hemorrhages and necrosis of mammary tissue. Major changes in teat and lactiferous sinuses and ducts; invasion of organism into parenchyma not a feature of E. coli.
Diagnostic confirmation. Culture of organism from milk and high SCC.
Other causes of acute and severe mastitis (must culture milk):
Treatment Must consider status and requirements for each case based on severity. Use of antimicrobial agents is indicated in moderately to severely affected animals; efficacy uncertain in mild cases. Some infections become persistent if antibiotics are not administered. Severely affected cattle also need supportive fluid and electrolyte therapy (such as hypertonic saline), and possibly NSAIDs for endotoxemia.
Control Manage outbreaks by examination of environment. Improve sanitation and hygiene. Regular cleaning of barns. Dry bedding. Avoid crowding. Keep dry cows on pasture if possible. Replace sawdust and shavings with sand for bedding. Emphasize premilking hygiene, including premilking germicide teat dipping and keep cows standing for at least 30 minutes after milking. Core lipopolysaccharide antigen vaccine in dry period and early lactation to reduce incidence of clinical mastitis due to Gram-negative bacteria.
The occurrence of coliform mastitis has increased considerably in recent years and is a cause for concern in the dairy industry and amongst dairy practitioners. Coliform mastitis occurs worldwide and is most common in dairy cattle that are housed in total confinement during the winter or summer months. Where cows are kept in total confinement in a drylot, outbreaks of coliform mastitis may occur during wet, heavy rainfall seasons. The disease is uncommon in dairy cattle that are continuously in pasture but it has been reported in pastured dairy cattle in New Zealand.
In contrast to contagious mastitis, environmental mastitis associated with coliform bacteria is primarily associated with clinical mastitis rather than subclinical mastitis. Clinical mastitis associated with environmental pathogens (including the environmental streptococci) is now the most important mastitis problem in well managed, low-SCC herds. In a survey of the incidence of clinical mastitis and distribution of pathogens in dairy herds in the Netherlands, the average annual incidence was 12.7 quarter cases per 100 cows per year. The most frequent isolates from clinical cases were E. coli (16.9%), S. aureus (14.4%), S. uberis (11.9%) and S. dysgalactiae (8.9%).1
The incidence of clinical coliform mastitis is highest early in lactation and decreases progressively as lactation advances.2 The rate of intramammary infection is about four times greater during the dry period than during lactation. The rate of infection is also higher during the first 2 weeks of the dry period and during the 2 weeks before calving. 80–90% of coliform infections results in varying degrees of clinical mastitis in the lactating cow; approximately 8–10% of coliform infections result in peracute mastitis, usually within a few days after calving. The disease also occurs commonly in herds that concentrate calving over a short period of time.
The prevalence of both intramammary infection and the incidence of clinical mastitis due to coliform bacteria has increased, particularly in dairy herds with a low prevalence of infection and incidence of clinical mastitis due to S. aureus and S. agalactiae as a result of an effective mastitis control program. Compared to other causes of mastitis, coliform infections are relatively uncommon and, in data based on herd surveys, the percentage of quarters infected with these pathogens is low. The percentage of quarters infected at any one time is generally low, at about 2–4%.
In the UK, about 0.2% of quarters of cows may be infected at any one time.3 Surveillance of a dairy herd in total confinement in the USA indicated that infection with coliform bacteria by either day of lactation or day of the year never exceeded 3.5% of quarters, and this maximum was reached on the day of calving. However, coliform infections may cause 30–40% of clinical mastitis episodes. In herds with a problem, up to 8% of cows have been infected with coliform bacteria, and 80% of the cases of clinical mastitis may be due to coliform infections.4
Coliform intramammary infections are usually of short duration. Over 50% last less than 10 days; about 70% less than 30 days; and only 1.5% exceed 100 days in duration.
The primary reservoir of coliform infection is the dairy cow’s environment (environmental pathogen); this is in contrast to the infected mammary gland, which is the reservoir of major contagious pathogens (S. aureus and S. agalactiae) and the main reservoir of infection in cattle with M. bovis. Exposure of uninfected quarters to environmental pathogens can occur at any time during the life of the cow, including during milking, between milkings, during the dry period and before calving in heifers.
In dairy herds with low bulk tank milk SCCs the average herd incidence of clinical mastitis is 45–50 cases per 100 cows annually, with coliforms isolated from 30–40% of the clinical cases. This is similar to an average incidence of 15–20 cases of coliform mastitis per 100 cows in herds with low bulk tank milk SCCs.5 Other observations indicate that the number of clinical cases of coliform mastitis varies from 3 to 32 per 100 cows per year but the average incidence in dairy herds can be as low as 6–8 per 100 cows per year.
Coliform mastitis is one of the most common causes of fatal mastitis in cattle. The case fatality rate from peracute coliform mastitis is commonly high and may reach 80% in spite of intensive therapy. Outbreaks of the disease can occur with up to 25% of recently calved cows affected within a few weeks of each other.
The isolates of E. coli from bovine mastitic milk are simply opportunist pathogens.6 The isolates that cause coliform mastitis possess lipopolysaccharides (endotoxin), which form part of the outer layer of the cell wall of all Gram-negative bacteria. Coliform bacteria isolated from the milk of cows or from their environment have different degrees of susceptibility to the bactericidal action of bovine sera, with almost all the isolates that cause severe mastitis being serum-resistant.7 Serum-sensitive organisms are unable to multiply in normal glands because of the activity of bactericidins reaching milk from the blood. Of the strains of E. coli isolated from cases of mastitis in cattle in England and Wales, only those that were serum-resistant were re-isolated from expressed milk following intramammary inoculation of lactating cows. Other observations indicate that serum-resistant coliforms have no selected advantage over serum-susceptible coliforms in naturally occurring intramammary infections. There are also somatic and capsular factors of coliforms that affect resistance to bovine bactericidal activity. Strains of Klebsiella that cause mastitis are also resistant to bovine serum. The fibronectin binding property of E. coli from bovine mastitis may be an important virulence factor that allows the organism to adhere to the ductular epithelium.
All the environmental components that come in contact with the udder of the cow are considered potential sources of the organisms. The coliform bacteria are opportunists, and contamination of the skin of the udder and teats occurs primarily between milkings when the cow is in contact with contaminated bedding rather than at the time of milking. Feces, which are a common source of E. coli, can contaminate the perineum and the udder directly or indirectly through bedding, calving stalls, drylot grounds, udder wash water, udder wash sponges and cloth rags, teat cups and milkers’ hands. Cows with chronic coliform mastitis also provide an important source of bacteria, and direct transmission probably occurs through the milking machine. Inadequate drying of the base of the udder and the teats after washing them prior to milking can lead to a drainage of coliform-contaminated water down into the teat cups and subsequent infection.
Sawdust and shavings used as bedding that are contaminated and harbor E. coli, and particularly K. pneumoniae, are major risk factors for coliform mastitis. Cows bedded on sawdust have the largest teat end population of total coliforms and klebsiellae; those bedded on shavings have an intermediate number and those on straw have the least. Experimentally, the incubation of bedding samples at 30–44°C (86–111°F) resulted in an increase in the coliform count; at 22°C (71°F) the count was maintained, and at 50°C (122°F) the bacteria were killed. Wet bedding, particularly sawdust and shavings, promotes the growth of coliform bacteria, especially Klebsiella spp.
The relationship between the bedding populations of Enterobacteriaceae was studied over a 12-month period in a dairy herd. The analyses revealed that rainfall bedding populations of E. coli and coliform mastitis incidence were statistically independent, while there was a strong association between rainfall and K. pneumoniae bedding populations and the incidence of K. pneumoniae mastitis. The lack of an association between bedding population of E. coli and coliform mastitis, along with the observation that cows are most susceptible immediately after parturition, suggest that the ability of the bacteria to penetrate the streak canal may be a factor of resistance in the cow and not a characteristic of the organism. Also, it appears that the cow in early lactation is not as susceptible to K. pneumoniae as to E. coli.
The ability of several different bedding materials to support the growth of environmental pathogens has been outlined under controlled conditions. Bedding materials vary in their ability to support growth of different pathogens, and under barn conditions it appears that high bacterial counts are influenced by factors more complex than type of bedding alone. Even clean damp bedding may support bacterial growth.
High populations of coliform bacteria on the teat end, unless accompanied by actual chronic quarter infection, are probably transitory and represent recent environmental contamination that would usually be eliminated by an effective sanitation program at milking time. However, any teat skin population, whether associated with infection in another quarter, from contaminated teat cup liners or from other environmental sources, must be considered as a potential source of new infection.
Factors that influence the susceptibility of cows to coliform mastitis include the SCC of the milk, the stage of lactation and the physiological characteristics and defense mechanisms of the udder (particularly the speed of neutrophil recruitment), teat characteristics, and the ability of the cow to counteract the effects of the endotoxins elaborated by the organisms.
Experimentally, an SCC of 250000 cells/mL in the milk of a quarter may limit significant growth of bacteria and development of mastitis when small inocula of coliform organisms are experimentally introduced into the gland.7 Somatic cell counts of 500000 cells/mL provided complete protection.8 Thus cows in herds with a low incidence of streptococcal and staphylococcal mastitis have a low milk SCC and are more susceptible to coliform mastitis. Dairy herds with low herd bulk tank milk SCCs may have a greater incidence of severe toxic mastitis than herds with higher counts.8
Increased susceptibility to coliform mastitis in the periparturient cow is primarily due to impaired neutrophil recruitment to the infected gland.9 In fatal cases of peracute mastitis in cows within 1 week after parturition there may be large numbers of bacteria in mammary tissues and an absence of neutrophilic infiltration. Other observations indicate a high correlation between poor preinfection chemotactic activity of blood neutrophils and susceptibility to intramammary E. coli challenge exposure.10 Experimentally, in periparturient cows the inability to recruit neutrophils rapidly into the mammary gland following intramammary infection is associated with an overwhelming bacterial infection and peracute highly fatal mastitis.9 The periparturient cows are unable to control bacterial growth during the first few hours after bacterial inoculation and consequently the bacterial load is much higher when neutrophils finally enter the milk. The lack of neutrophil mobilization could be due to:
• Failure to recognize bacteria
• Lack of production of inflammatory mediators
• A defect in the ability of the cells to move into the milk compartment.
In ketonemic cows, experimental E. coli mastitis is severe, regardless of preinfection chemotactic response.11
High levels of cytokines are present in the milk of cows that lack the ability to recruit leukocytes, which is evidence that the cells recognized the bacteria.9 All of this suggests that the critical defect is in the neutrophils of the periparturient cow. Certain cell-surface receptors on leukocytes may be important defense mechanisms against E. coli polysaccharides.12 Bovine mammary neutrophils possess cell surface C14 and C18 and lectin– carbohydrate interactions mediating non-opsonic phagocytosis of E. coli, which may be important in controlling these infections.
The positive effects of supplemental vitamin E and selenium on mammary gland health are well established. An adequate dietary level of selenium enhances the resistance of the bovine mammary gland to infectious agents. Experimentally induced intramammary E. coli infections are significantly more severe, and of longer duration, in cows whose diets have been deficient in selenium than in cows whose diets were supplemented with selenium. The enhanced resistance is thought to be associated with a more rapid diapedesis of neutrophils into the gland of cows fed diets supplemented with selenium, which limits the numbers of bacteria in the gland during infection.
Vitamin E is especially important to mammary gland health during the peripartum period. Plasma concentrations of alpha-tocopherol begin to decline at 7–10 days before parturition, reach nadir at 3–5 days after calving and then start increasing.13 When plasma concentrations are maintained during the peripartum period by injections of alpha-tocopherol, the killing ability of blood neutrophils is improved.14 The supplementation of the diets of dry cows receiving 0.1 ppm of selenium in their diets with vitamin E at 1000 IU/d reduced the incidence of clinical mastitis by 30% compared to cows receiving 100 IU/d. The reduction was 88% when cows were fed 4000 IU/d of vitamin E during the last 14 days of the dry period.2
There are also marked effects of dietary selenium on milk eicosanoid concentrations in response to an E. coli infection, which may be associated with the altered pathogenesis and outcome of mastitis in a selenium-deficient state.15
Coliform mastitis occurs almost entirely in the lactating cow and rarely in the dry cow. The disease can be produced experimentally in lactating quarters much more readily than in dry quarters.7 The difference in the susceptibility may be due to the much higher SCCs and lactoferrin concentrations in the secretion of dry quarters than in the milk of lactating quarters. Cows with known uninfected quarters at drying off may develop peracute coliform mastitis at calving, suggesting that infection occurred during the dry period. New intramammary infections can occur during the nonlactating period, especially during the last 30 days, remain latent until parturition and cause peracute mastitis after parturition.
The rate of coliform intramammary infection is highest during the 2 weeks following drying off and in the 2 weeks before calving. The fully involuted mammary gland appears to be highly resistant to experimental challenge by E. coli but it becomes susceptible during the immediate prepartum period. More than 93% of E. coli intramammary infection associated with the nonlactating period originated during the second half of that period.16
Several physiological factors may influence the level of resistance of the nonlactating gland to coliform infection. The rate of new intramammary infection is highest during transitions of the mammary gland from lactation to involution and during the period of colostrum production to lactation. There can be a sixfold increase in coliform infections from late lactation to early involution but 50% of these new infections do not persist into the next lactation. Also, the rate of spontaneous elimination of minor pathogens is high during the nonlactating period. The difference in susceptibility or resistance to new intramammary infection may be due, in part, to changes in concentration of lactoferrin, IgG, bovine serum albumin and citrate, which are correlated with in vitro growth inhibition of K. pneumoniae, E. coli, and S. uberis.
There is also a slower increase in polymorphonuclear neutrophils in milk after new intramammary infection in early lactation than in mid and late lactation. These conditions may explain the occurrence of peracute coliform mastitis in early lactation. This suggests latent infection or, more likely, that infection occurred at a critical time just a few days before and after calving, when the streak canal became patent and the population of coliform bacteria on the teat end was persistently high because the cow was not being milked routinely and thus would not be subjected to udder washing and teat dipping. Coliform bacteria can pass through the streak canal unaided by machine milking – this may be associated with the high incidence of coliform mastitis in high-yielding older cows, which may have increased patency of the streak canal with age.
Newly calved cows can be classified as moderate or severe responders to experimentally induced coliform mastitis. Following infection there is a diversity of responses varying from very mild to very acute inflammation of the gland and evidence of systemic effects such as fever, anorexia and discomfort.17 Losses in milk yield and compositional changes are most pronounced in inflamed glands and, in severe responders; milk yield and composition did not return to preinfection levels. It is proposed that the severe and long-lasting systemic disturbances in severe responders can be attributed to absorption of endotoxin.
In summary, coliform mastitis is more severe in periparturient cows because of inability to slow bacterial growth early after infection. This inability is associated with low SCC before challenge and slow recruitment of neutrophils.9 There may also be deficits in the ability of leukocytes to kill bacterial pathogens.
The sporadic occurrence of the disease may be associated with the use of contaminated teat siphons and mastitis tubes and infection following traumatic injury to teats or following teat surgery. Several teat factors are important in the epidemiology of E. coli mastitis. It is generally accepted that E. coli is common in the environment of housed dairy cows and that mastitis can be produced experimentally by the introduction of as few as 20 organisms into the teat cistern via the teat duct. However, the processes by which this occurs under natural conditions are unknown. E. coli does not colonize the healthy skin of the udder or the teat duct.
The teat duct normally provides an effective barrier to invasion of the mammary gland by bacteria. As a result of machine milking there is some relaxation of the papillary duct, followed by gradual reduction in the duct lumen diameter in the 2 hours following milking. This period of relaxation after milking may be a risk factor predisposing to new intramammary infection.
Experimental contamination of the teat ends with a high concentration of coliform bacteria by repeated wet contact, however, does not necessarily result in an increase in new intramammary infection. The experimental application of high levels of teat end contamination with E. coli after milking repeatedly led to high rates of intramammary infection, which suggests that penetration of the teat duct by E. coli occurs in the period between contamination and milking. Milking machines that produce cyclic and irregular vacuum fluctuations during milking can result in impacts of milk against the teat ends, which may propel bacteria through the streak canal and increase the rate of new infections due to E. coli and outbreaks of peracute coliform mastitis.
Cows affected with the downer cow syndrome following parturient paresis, or recently calved cows that are clinically recumbent for any reason, are susceptible to coliform mastitis because of the gross contamination of the udder and teats with feces and bedding.
The failure of lactoferrin within mammary secretions to prevent new infections and mastitis near and after parturition may be due to a decrease in lactoferrin before parturition. Lactoferrin normally binds the iron needed by iron-dependent organisms; these multiply excessively in the absence of lactoferrin. Also, citrate concentrations increase in mammary secretions at parturition and may interfere with iron-binding by lactoferrin.
The serum IgG1 ELISA titers recognizing core lipopolysaccharide antigens of E. coli J5 in cattle are associated with a risk of clinical coliform mastitis. Titers less than 1:240 were associated with 5.3 times the risk of clinical coliform mastitis. Older cattle in the fourth or greater lactations were also at greater risk, even though titers increased with age. There is a titer-independent age-related increase in clinical coliform mastitis. Active immunization of cattle with an Rc-mutant E. coli (J5) vaccine resulted in a remarkable decrease in the incidence of clinical coliform mastitis.18
After invasion and infection of the mammary gland, E. coli proliferates in large numbers and releases endotoxin on bacterial death or during rapid growth when excess bacterial cell wall is produced. Endotoxin causes a change in vascular permeability, resulting in edema and acute swelling of the gland and a marked increase in the number of neutrophils in the milk.7 The neutrophil concentrations may increase 40–250 times and strongly inhibit the survival of E. coli.7 This marked diapedesis of neutrophils is associated with the remarkable systemic leukopenia and neutropenia that occurs in peracute coliform mastitis. The severity of the disease is influenced by:
• The degree of the pre-existing neutrophils in the milk
• The rate of invasion and total number of neutrophils that invade the infected gland
• The susceptibility of the bacteria to serum bactericidins that are secreted into the gland
• The amount of endotoxin produced.19
The severity of disease is dependent on the stage of lactation. Experimental infection of the mammary gland of recently calved cows with E. coli produces a more severe mastitis when compared with animals in midlactation. This may be due to a delay in diapedesis of neutrophils into the mammary gland of recently calved cows. Furthermore, because of this delay there may be no visible changes in the milk for up to 15 hours after infection, but the systemic effects of the endotoxin released by the bacteria are evident in the cow (fever, tachycardia, anorexia, rumen hypomotility or atony, mild diarrhea). The net result is endotoxemia, which persists as long as bacteria are multiplying and releasing endotoxin. This persistent endotoxemia is probably a major cause of failure to respond to therapy compared to the transient endotoxemia in the experimental inoculation of one dose of endotoxin.
The final outcome is highly dependent on the degree of neutrophil response.20 If the neutrophil response is delayed and growth of the organisms is unrestricted, the high levels of toxin produced could cause severe destruction of udder tissue and general toxemia. If the animal responds quickly there is often little effect on milk yield because the injury is confined to the sinuses without involvement of secretory tissues.7 The ability of the neutrophils to kill E. coli varies among cows. Experimental infection of the mammary gland of cows with E. coli results in the stimulation of a long-lasting opsonic activity for the phagocytosis and killing of the homologous strain of the organism by neutrophils. Thus it is not opsonic deficiency that is the problem in early lactation but rather a failure of rapid migration of neutrophils into the gland cistern.
The rapidity and efficiency of the neutrophil response are major factors in determining the outcome.21 If the neutrophil response is rapid, clinical disease will be mild or go undetected, self-cure will occur and the cow returns to normal; the milk may be negative for the bacteria. This may be one important cause of an increase in the percentage of clinical mastitis cases in which no pathogens can be isolated from the milk. Failure of the cow to mount a significant neutrophil response results in the multiplication of large numbers of bacteria, the elaboration of large amounts of endotoxin and severe highly fatal toxemia. In these cases, bacteria are readily cultured from the milk. In less serious and nonfatal cases, the recruitment of neutrophils does not fail but is delayed; this results in acute clinical mastitis with progressive inflammation and permanent loss of secretory function. The bacteria are not always readily eliminated from the infected gland by the neutrophils. Coliform bacteria may remain latent in neutrophils and, in naturally occurring cases, it is not uncommon to be able to culture the organism from the mammary gland during and after both parenteral and intramammary antibacterial therapy.
The numbers of bacteria in the milk also influence the outcome. If bacterial numbers exceed 106 cfu/mL, the ability of the neutrophil to phagocytose is impaired. If the bacterial count is less than 103 cfu/mL at 12 hours postinfection, the bacteria will be rapidly eliminated and the prognosis will be favorable. This response is seen as a subacute form of the disease with spontaneous self-cure. If the neutrophil response is slow or delayed, the cow will exhibit more severe signs of coliform mastitis due to toxemia. This is most common in recently calved cows and is characterized clinically by a serous secretion in the affected quarter that later becomes watery, fever, depression, ruminal hypomotility and mild diarrhea. The prognosis in these cases is unfavorable. These more severe forms of coliform mastitis usually occur after calving and in the first 6 weeks of lactation. Cows with coliform mastitis in mid to late lactation generally generate a rapid neutrophil response rate and their prognosis is likely to be favorable.
In an attempt to further understand the pathogenesis of coliform mastitis, the effect of experimentally introducing E. coli endotoxin into the mammary gland has been examined. The intramammary infusion of 1 mg E. coli endotoxin induces acute mammary inflammation and transient, severe shock from which cows recover within 48–72 hours.22 Udder edema is apparent within 2 hours but begins to subside in 4–6 hours. The SCC increases within 3–5 hours and at 7 hours the count is 10 times normal. A mild systemic reaction with a transient fever occurs in some cows. High concentrations of interleukin-1 and interleukin-6 are detectable in the milk of infused glands, beginning 3–4 hours after infusion.23 Milk concentrations of bovine serum albumin are increased from baseline levels to peak levels within 2 hours, indicating increased vascular permeability induced by inflammatory mediators. The infusion of endotoxin into the teat cistern of cows induces a rapid local inflammatory response of short duration with an influx of neutrophils into the teat cistern.24
The intramammary infusion of endotoxin results in a sequential increase of immunoglobulin in milk whey and of phagocytosis of staphylococci by milk polymorphonuclear cells. This is consistent with spontaneous recovery of cows with acute coliform mastitis. Endotoxin infusion can also result in increases in arachidonic acid metabolites such as thromboxanes, and cytokines,23 which may be involved in mediation of local quarter inflammation and the systemic signs observed in acute coliform mastitis. Histamine, serotonin, leukotrienes and arachidonic metabolites are also released following experimental K. pneumoniae mastitis. There is also a marked increase in prostaglandin concentrations, which indicates that they may play a role in the pathogenesis of endotoxin-induced mastitis and that the use of NSAIDs may be of value therapeutically.
In peracute coliform mastitis, severe toxemia with fever, shivering, weakness leading to recumbency in a few hours, and mild diarrhea are common and probably due to the absorption of large quantities of endotoxin. For many years it was thought that bacteremia did not occur in severe cases of coliform mastitis. However, bacteremia is present in 32–48% of naturally occurring cases of coliform mastitis.25,26 In experimental endotoxemia in cattle there is profound leukopenia (neutropenia and lymphopenia), a mild hypocalcemia and elevation in plasma cortisol concentration. Hypocalcemia also occurs in naturally occurring cases and is thought to be due to decreased abomasal emptying rate associated with the endotoxemia. Experimentally infused endotoxin is detoxified very rapidly after absorption into the circulation.
In the acute form, the systemic changes are usually less severe than in the peracute form. However, in both forms, there is marked agalactia and the secretions in the affected quarter become serous and contain small flakes. Coliform organisms are not active tissue invaders, and in affected cattle that survive the systemic effects of the endotoxin the affected quarter(s) will usually return to partial production in the same lactation, and even full production in the next. However, in some cows that survive the peracute form, subsequent milk production in the current lactation is inadequate and cows are commonly culled.
A retrospective analysis of cows with clinical and laboratory features of coliform mastitis revealed that 60% returned to produce a milk-like secretion in the affected quarters in the current lactation and 40% did not. However, only 63% of the former group and 14% of the latter group remained in the herd and produced milk in the next lactation. Some cows were culled during the current lactation for low milk production and other reasons, some died and others were culled for mastitis. Of the original 88 cows with coliform mastitis, only 38 (43%) remained in the herd and produced milk in the next lactation.27
Peracute coliform mastitis in the cow is a severe disease characterized by a sudden onset of agalactia and toxemia. The cow may be normal at one milking and acutely ill at the next. Complete anorexia, severe depression, shivering and trembling, cold extremities (particularly the ears) and a fever of 40–42°C (104–108°F) are common. Within 6–8 hours after the onset of signs the cow may be recumbent and unable to stand. At that stage, the temperature may be normal or subnormal, all of which may superficially resemble parturient paresis. The heart rate is usually increased up to 100–120 beats/min, the rumen is static, there may be a mild watery diarrhea and dehydration is evident. Polypnea is common and in severe cases an expiratory grunt may be audible because of pulmonary congestion and edema.
The affected quarter(s) is usually swollen and warm but not remarkably so, and for this reason coliform mastitis may be missed on initial clinical examination. The cow may be severely toxemic, febrile and have cold extremities before there are visible changes in the mammary gland or the milk. The mammary secretion is characteristic, and changes from the consistency of watery milk initially to a thin, yellow serous fluid containing small meal-like flakes that are barely visible to the naked eye and are best seen on a black strip plate used for gross examination of milk. Additional quarters may become affected within a day or two of the initial infection.
The course of peracute coliform mastitis is rapid. Some cows will die in 6–8 hours after the onset of signs; others will live for 24–48 hours. Those that survive the peracute crisis will either return to normal in a few days or remain weak and recumbent for several days and eventually develop the complications associated with prolonged recumbency. Intensive intravenous fluid therapy may prolong the life of the cow for up to several days but significant improvement may not occur and eventually euthanasia may appear to be the desirable course of action.
Acute coliform mastitis is characterized by varying degrees of swelling of the affected gland and variable systemic signs of fever and inappetence. The secretions of the gland are watery to serous in consistency and contain flakes. Recovery with appropriate treatment usually occurs in a few days.
Various diagnostic schemes that use clinical parameters to differentiate cows with clinical mastitis due to Gram-negative bacteria from those with clinical mastitis associated with Gram-positive bacteria have been developed.28-34 In general, all these schemes predict Gram-negative bacteria as the cause if the milk is watery or yellow, if the mastitis episode occurs in summer and if rumen motility is decreased or absent. Experienced clinicians are not much better at predicting the causative agent than inexperienced clinicians. The conclusion from all of these studies is that clinical observations do not allow sufficiently accurate prediction of clinical mastitis pathogens and should not be used as the sole criteria for deciding whether cows are treated with antibiotics, or even the class of antibiotic to be administered.34 Even the best predictive algorithm was wrong 25% of the time if the prevalence of Gram-negative mastitis was 50%, which is too high an error rate to be used to guide treatment. For comparison, flipping a coin to attribute the causative agent as being Gram-positive or Gram-negative is wrong only 50% of the time!
An increase in the ability of a positive test to predict a Gram-negative bacterial infection as the cause for a clinical mastitis episode is provided by examining for the presence of endotoxin in milk (sensitivity (Se) = 0.72; specificity (Sp) = 0.95),35 whether the segmented neutrophil count is less than 35% of the total leukocyte count (Se = 0.87; Sp = 0.71),36 whether the segmented neutrophil count is less than 3200 cells/μL (Se = 0.93; Sp = 0.89),36 and by culturing on selective media (Se = 0.60; Sp = 0.98).37 The endotoxin test is a cowside test (Limast-test®) that is commercially available in Scandinavia. The test takes 15 minutes to run on milk samples and requires at least 104–105 cfu of Gram-negative bacteria for a positive test result.35 Assessment of the white blood cell count and differential count is widely available in veterinary practice but is not a cowside test and is therefore not ideal. Both the milk endotoxin test and blood neutrophil count have adequate sensitivity and specificity for use to guide treatment decisions.
Chronic coliform mastitis is characterized by repeated episodes of subacute mastitis, which cannot be readily clinically distinguished from other common causes of mastitis.
Subclinical coliform mastitis is characterized by the presence of coliform organisms in the milk samples of cows without clinical evidence of mastitis. The prevalence of intramammary infection in quarters with coliform bacteria is low relative to contagious mastitis pathogens, ranging from 0.9–1.2%.
Milk samples should be submitted for culture to identify the causative agent, but antimicrobial susceptibility testing has not been validated and is currently not recommended to guide treatment decisions.38 In the peracute case, the milk samples will yield a positive culture. In less acute cases, the milk sample may be negative because the neutrophils have cleared the bacteria.
In the experimental disease the SCC of milk from the inoculated quarter ranges from 14000000–25000000 cells/mL at 5 hours after inoculation. The CMT on secretions from affected quarters is usually +3.
In peracute coliform mastitis there is hemoconcentration, a marked leukopenia, neutropenia and a degenerative left shift due to the margination of large numbers of neutrophils in response to endotoxin. There is also a moderate lymphopenia, monocytopenia and thrombocytopenia.36 If the degenerative left shift, leukopenia and neutropenia become worse on the second day after the onset of clinical signs, the prognosis is unfavorable. An improvement in the differential white count on the second day is a good prognostic sign.
A commercially available cowside test (Limast-test®) for endotoxin is available in Scandinavia. The test takes 15 minutes to run on milk samples and is able to detect the presence of endotoxin and therefore Gram-negative bacteria, but does not differentiate between E. coli and K. pneumoniae.
The biochemical abnormalities observed in naturally occurring cases include uremia, high aspartate aminotransferase activity and strong ion (metabolic) acidosis in fatal cases, while in surviving cases there were decreased concentrations of sodium, potassium and chloride, and strong ion (metabolic) alkalosis.25,36
There is edema and hyperemia of the mammary tissue. In severe cases hemorrhages are present and are accompanied by thrombus formation in the blood and lymphatic vessels; there is necrosis of the parenchyma.
A study of the progressive pathological changes in experimental and natural cases of E. coli mastitis in cows reveals that damage is most marked in the epithelium of the teat and lactiferous sinuses and diminishes rapidly towards the ducts. In hyperacute cases, the organisms are largely confined to the ductular and secretory lumen and there is little invasion of the parenchyma, despite the presence of large numbers of organisms. In some cases there may be intense neutrophil infiltration, subepithelial edema and epithelial hyperplasia of the sinuses and large ducts. In hyperacute cases in the immediate postpartum period, infiltration of neutrophils may be negligible. There is now some evidence that bacteremia may occur in coliform mastitis.25
• Peracute coliform mastitis in cattle is characterized clinically by a sudden onset of toxemia, weakness, shivering, often recumbency, fever in the early stages followed by a normal temperature or hypothermia in several hours, and characteristic gross changes in the milk, which usually is watery and contains some particles barely visible to the unaided eye. The peracute form of the disease is most common in recently calved cows
• Parturient hypocalcemia paresis occurs in recently calved cows. The weakness and recumbency resembles peracute coliform mastitis but the marked increase in heart rate, and dehydration and mild diarrhea if present, are not characteristic of parturient paresis and should prompt further clinical examination, particularly of the udder. In the early stages of coliform mastitis the changes in the milk may be just barely visible. Those clinical findings which are most useful to predict peracute coliform mastitis include watery consistency of milk, shivering, firmness of udder, tachycardia, polypnea, fever, weakness and mastitis of less than 24 hours’ duration.31 A marked leukopenia and neutropenia are characteristic of coliform mastitis, whereas in parturient paresis there is usually a neutrophilia and stress leukon (neutrophilia, no left shift, lymphopenia, monocytosis and eosinopenia). The differential diagnosis of recumbency in the immediate postpartum period is discussed under parturient paresis
• Carbohydrate engorgement lactic acidosis causes rapid onset of weakness, recumbency, diarrhea, dehydration, and ruminal stasis and resembles the clinical findings of shock in peracute coliform mastitis. However, the rumen contains an excess of watery fluid and the pH is below 5
• Acute coliform mastitis cannot be accurately differentiated from all other common causes of acute mastitis with abnormal gland and abnormal milk, including the environmental streptococci S. uberis and S. dysgalactiae, and the contagious pathogens S. aureus and S. agalactiae. Culture of the milk is necessary
The treatment of coliform mastitis in cattle has been controversial but recent studies have clarified the important role that antimicrobial agents play in treating severely affected cattle. Historically, the treatment of coliform mastitis was based on the principles of treating a bacterial infection with varying degrees of inflammation. A combination of broad-spectrum antimicrobial agents administered parenterally and by intramammary infusion, fluid and electrolyte therapy, frequent stripping out of the affected glands with the aid of oxytocin, and anti-inflammatory drugs have been used with varying degrees of success based on empirical and anecdotal experience. Only a handful of clinical trials have evaluated the efficacy of therapeutic agents used in naturally occurring cases of coliform mastitis, especially for the peracute form of the disease.
Most of the controversy has centered on the rational use of antimicrobial agents.4 The use of antimicrobial agents for the treatment of coliform mastitis has been questioned for several reasons:
• Clinical signs are primarily due to the effects of endotoxin in the mammary gland, with formation of endogenous inflammatory mediators within the udder and their subsequent release into the systemic circulation39
• The severity of clinical signs are correlated with the number of bacteria in the affected gland
• Most mild cases of coliform mastitis (abnormal milk but normal gland and cow) are self-limiting and resolve without antimicrobial therapy. However, a small percentage of these mild clinical cases develop persistent infection
• There is speculation that the use of bactericidal antimicrobial agents may result in the bolus release of large quantities of lipopolysaccharides in the mammary gland associated with a rapid kill of bacteria, but this has not been observed in any study to date. In contrast, endotoxin release occurs from rapid bacterial growth alone, which will be prevented by administration of an effective antibiotic
• Many, but not all, of the broad-spectrum antimicrobial agents currently approved for use in lactating cattle do not result in high enough concentrations in the milk when given parenterally.
Most of the antimicrobial agents currently used for the treatment of coliform mastitis in lactating dairy cows are not approved for use in food-producing animals.40 Because of this extralabel use and the lack of pharmacokinetic data for adequate withholding times, the risk of drug residues in milk and meat is increased.
The prognosis in the peracute form of the disease is unfavorable if severe clinical toxemia is present. Severe depression, weakness, diarrhea and dehydration, recumbency and a heart rate over 120 beats/min are indicators of an unfavorable prognosis. The successful treatment of peracute coliform mastitis requires the earliest possible action and clinical surveillance until recovery is apparent.
Treatment trials with experimentally induced coliform mastitis in cattle during lactation have failed, for the most part, to demonstrate efficacy of antimicrobial therapy. This is because all experimental models used to date do not accurately reproduce the naturally occurring disease,41,42 and not because antibiotics are ineffective. Accordingly, treatment efficacy should be based on the results of randomized field trials. The major considerations for antimicrobial use in coliform mastitis include:
• Early administration in order to decrease the exposure of the cow to endotoxin
• Ensuring appropriate withholding periods for milk and meat
• The benefit–cost ratio.5
The antimicrobial susceptibilities of E. coli isolates from coliform mastitis vary considerably; drug susceptibility determination is not routinely recommended because the breakpoints have not been validated and the bacteria come from diverse sources in the environment.
Broad-spectrum antimicrobial agents should be administered parenterally to cattle with systemic signs of disease (abnormal cow), preferably by the intravenous route initially, followed by intramuscular administration to maintain appropriate plasma concentrations. The first reason to administer parenteral antibiotics is that the severity of clinical signs is correlated with the numbers of bacteria in milk from the affected gland.43 The second main reason to administer parenteral antibiotics is to combat bacteremia, which is present in 32–48% of severely affected cattle.25,26,44 Based on pharmacokinetic/pharmacodynamic values, the results of experimentally induced and naturally acquired infections, and in vitro antimicrobial susceptibility testing (if this has any relevance to in vivo susceptibility), most E. coli isolated from the mammary glands of cattle are theoretically susceptible to third-generation cephalosporins (such as ceftiofur), fourth-generation cephalosporins (such as cefquinome), fluoroquinolones, gentamicin, amikacin, trimethoprim–sulfonamide and oxytetracycline.
• Ceftiofur is a third-generation cephalosporin that is resistant to beta-lactamases and has excellent in vitro activity against E. coli. When given parenterally to cows with experimental coliform mastitis, ceftiofur did not produce drug concentrations in milk above the reported minimum inhibitory concentrations for coliform bacteria.45 However, when administered to cows with naturally occurring coliform mastitis, ceftiofur-treated cows (2.2 mg/kg BW intramuscularly every 24 h) were three times less likely to die or be culled from the herd and had more saleable milk than nontreated cattle44
• Cefquinome is a fourth-generation cephalosporin that is resistant to beta-lactamases and has excellent in vitro activity against E. coli. Parenteral cefquinome therapy (1 mg/kg BW intramuscularly twice at 24 h apart), with or without intramammary cefquinome (75 mg, three times at 12 h intervals), increased the bacteriological cure rate and significantly improved clinical recovery and return to milk production in experimentally induced E. coli mastitis46
• Enrofloxacin, a fluoroquinolone with excellent in vitro activity against E. coli, given intravenously initially then subcutaneously (5 mg/kg BW) was effective in treating experimentally induced E. coli mastitis.47-49 In general, parenterally administered enrofloxacin increased the rate of E. coli clearance from the infected mammary gland
• Gentamicin has been used on a extralabel basis for the treatment of acute and peracute coliform mastitis because more than 90% of isolates from milk from affected cows are sensitive in vitro.40 However, the parenteral administration of gentamicin at 2 g intramuscularly every 12 hours until the appetite improved to dairy cows with mastitis predicted to be associated with Gram-negative bacteria did not result in significant improvement compared to cows with similar mastitis that did not receive an antimicrobial or received erythromycin19
• Trimethoprim–sulfadiazine (trimethoprim 4 g, sulfadiazine 20 g, intramuscularly every 24 h for 3–5 d) is efficacious in treating naturally acquired cases of coliform mastitis. The recovery rate of cows with clinical mastitis due to coliform bacteria susceptible to sulfonamide– trimethoprim was 89% compared to 74% in cows infected with coliforms resistant to the combination given parenterally, combined with NSAIDs and complete milking of affected quarters several times daily.50 Sulfadiazine or sulfamethazine (sulfadimidine) are preferred to sulfadoxine because the latter produces much lower milk concentrations after parenteral administration
• Oxytetracycline (16.5 mg/kg BW intravenously every 24 hours for 3–5 d), combined with intramammary cephapirin (200 mg) and supportive care (intravenous or oral fluids, flunixin meglumine, stripping of the mammary gland) was more effective in treating coliform mastitis than similar treatment without antibiotics in cattle with naturally acquired mastitis.
Intramammary preparations of antimicrobial agents can be infused into the affected quarters after they have been stripped out completely at the start and end of the day. The initial choice of antimicrobial will depend on previous experience of treatment efficacy in the herd.
• Ceftiofur: based on clinical response and the results of antimicrobial susceptibility testing of coliform isolates from cows with naturally occurring mastitis (if relevant to in vivo performance), ceftiofur is an excellent choice for intramammary infusion in suspected cases of coliform mastitis51
• Gentamicin: the intramammary infusion of 500 mg of gentamicin did not affect the duration or severity of experimentally induced coliform mastitis.40 The numbers of E. coli in the milk after intramammary inoculation were not affected by the intramammary infusion of gentamicin, despite maintaining a mean minimal gentamicin concentration in milk of 181 μg/mL between dose intervals.40 The infusion did not affect the body temperature or the magnitude and duration of the inflammatory process in the glands as measured by the SCCs and peak albumin and immunoglobulin concentrations in the milk. It should be noted that gentamicin is not approved for use in the treatment of bovine mastitis and in some jurisdictions is not approved for any use.
A study of the efficacy of intramammary antibiotic therapy for the treatment of naturally occurring clinical mastitis associated with environmental pathogens found no difference in the short-term clinical or bacteriological cure rates between quarters infused with 62.5 g amoxicillin every 12 hours for three milkings or 200 mg of cephapirin every 12 hours for two milkings, and those treated with 100 units of oxytocin intramuscularly every 12 hours immediately before milking for two or three milkings alone.52 However, the cost per episode of mastitis associated with the use of cephapirin was higher than the other two treatments, partly because of the longer milk withdrawal time (96 h) associated with the drug. The percentage of relapses was higher for cows in the oxytocin treatment group, especially when the mastitis-associated pathogen was an environmental Streptococcus sp.53
An artificial intramammary environment has shown that milking 12 times daily could lead to elimination of E. coli,54 suggesting that frequent stripping would be an effective treatment. Indeed, stripping (augmented by oxytocin) is a popular but largely unsubstantiated recommendation for treating severe cases of coliform mastitis.
Oxytocin at 10–20 units per adult cow given intramuscularly, followed by vigorous hand massage and hourly stripping of the affected quarter, may assist in removing inflammatory debris. Oxytocin doses higher than this are not needed, and intravenous administration is not needed because oxytocin is rapidly absorbed when injected intramuscularly. Oxytocin can be repeated and used as long as an effect is obtained.
Effective removal of coliform bacteria and endotoxin will minimize their local effects in the mammary gland and decrease the systemic signs of endotoxemia. The main problems with stripping are the labor involved, the small volumes produced, the potential for creating additional pain and discomfort for the cow (and the producer when the cow kicks!) and potential contamination of the environment if the secretion is stripped on to the ground. The role of frequent stripping, if any, in the treatment of clinical mastitis remains to be determined.
Fluid and electrolyte therapy are essential for the treatment of acute and peracute coliform mastitis in order to counteract the effects of the endotoxemia. Isotonic polyionic electrolyte solutions (such as Ringer’s solution) are given at 80 mL/kg BW for the first 24 hours by continuous intravenous infusion, and at a slower rate than that over the following days. For a mature dairy cow (400–600 kg) a total of 32–48 L is therefore needed in the first 24-hour period, with 20 L given during the first 4 hours and the remainder over the next 20 hours. A favorable response is usually clinically evident in 6–8 hours. If the animal has not improved after 5 days of intensive fluid therapy (the 5-day rule for clinical improvement), the prognosis for survival is poor.
The large amounts of isotonic fluids and electrolytes which have been advocated and used are expensive to administer by continuous intravenous infusion and require monitoring over many hours. A possible alternative is the use of small volumes of hypertonic saline, which can be transported easily and administered rapidly. Hypertonic saline can be safely administered to cattle with endotoxin-induced mastitis.55 Hypertonic saline (7.2% NaCl) is given intravenously at 4–5 mL/kg BW intravenously over 4–5 minutes followed by immediate access to drinking water.56 The changes following administration of hypertonic saline include transient expansion of the plasma volume, hypernatremia and hyperchloremia.56 The intravenous administration of hypertonic saline to clinically normal cows with access to water increases circulatory volume rapidly, induces slight strong ion (metabolic) acidosis and increases glomerular filtration rate.57 Fluid therapy is covered in detail in Chapter 2.
NSAIDs are frequently administered as adjunctive therapy in coliform mastitis, particularly in the peracute form of the disease.39 Ketoprofen is the only currently available NSAID with documented efficacy in naturally acquired cases of coliform mastitis.
Ketoprofen has been evaluated as adjunctive therapy for the treatment of acute clinical mastitis in dairy cows, most cases of which were associated with Gram-negative pathogens.58 All cases were treated with 20 g sulfadiazine and 4 g trimethoprim intramuscularly followed by one-half dose daily until recovery. Ketoprofen was given at 2 g intramuscularly daily for the duration of the antimicrobial therapy. Recovery rates for the nonblind contemporary controls and the blind placebo controls were 84% and 71%, respectively. In the nonblind controlled ketoprofen and placebo-controlled ketoprofen treatment groups, recovery rates were 95% and 92%, respectively. The odds ratio (OR) of recovery was significantly high in the placebo-controlled study (OR = 6.8), and high but not significant in the nonblind controlled study (OR = 2.6). It was concluded that ketoprofen significantly improved recovery rate in clinical mastitis. A similar clinical field trial evaluating the efficacy of phenylbutazone and dipyrone for the treatment of mastitis caused mostly by coliforms revealed a beneficial effect but no difference between the efficacies of the two drugs.59 Neither phenylbutazone nor dipyrone is permitted for use in lactating dairy cattle in the USA, but their use is permitted in some countries.
The anti-inflammatory effect of either flunixin meglumine or dexamethasone was evaluated compared to controls in experimentally induced coliform mastitis. Dexamethasone at 0.44 mg/kg intravenously and flunixin meglumine at 1.1 mg/kg intravenously were both given 2 hours after inoculation of the E. coli, which is essentially a pretreatment administration because clinical signs are not evident at this time. Flunixin meglumine was also administered once 8 hours after the initial dose. Dexamethasone reduced the rectal temperature and the mammary surface temperatures, and prevented further increase in rectal temperature above 39.2°C. The response to flunixin meglumine was less than expected, which suggested that a higher dose of 2.2 mg/kg may be necessary in lactating dairy cattle. The administration of flunixin meglumine at 2.2 mg/kg intramuscularly or flurbiprofen at 2 mg/kg intravenously before clinical signs appeared in experimental E. coli mastitis abolished the febrile response during the first 9 hours after infection and lessened the decrease in rumen motility. Carprofen, a long-acting NSAID, reduced the fever, tachycardia and udder swelling associated with E. coli-endotoxin-induced mastitis.60 The long-acting properties of carprofen may be considered a therapeutic advantage over flunixin meglumine, which requires frequent dosing.
Fluid and electrolyte therapy and flunixin meglumine, in combination and individually, have been evaluated in a 3-year study of a large number of cows with toxic mastitis.61 Cows were allotted to one of three groups:
• Fluid therapy (45 L of intravenous isotonic electrolyte solution) and flunixin meglumine at 2 g
All cases were treated with parenteral and intramammary antimicrobial agents, oxytocin and calcium borogluconate. There was no significant difference in the rate of survival between the treatment groups, and 54% of the cows survived.
The control of coliform mastitis is characteristically difficult, unreliable and frustrating. Several cases of fatal peracute coliform mastitis may occur in a herd of 100 cows during a period of a year, in spite of the existence of apparently excellent management. The general principles of mastitis control that have been effective for the control of S. aureus and S. agalactiae mastitis have been unsuccessful for the control of coliform mastitis because infection of the mammary gland occurs by direct contact with the environment, usually between milkings. For the control of coliform mastitis, the emphasis is on the prevention of new infection. Core lipopolysaccharide antigen vaccines are useful and are discussed below.
When an outbreak of peracute coliform mastitis is encountered the following procedures are recommended in an attempt to prevent new cases:
• Culture milk samples and obtain a definitive etiological diagnosis (in other words, put a name to the causative pathogen)
• Examine the bedding for evidence of heavy contamination with coliform bacteria. If sawdust or wood shavings are being used, replace with sand, if possible, or change more frequently
• Conduct a general clean-up of the stall and lounging areas
• Examine milking machine function
• Allow cows access to fresh feed immediately after milking to ensure that they remain standing for at least 30 minutes to allow time for the streak canal to close.
The normal presence of coliform bacteria in every aspect of the cow’s environment must be recognized but every effort must be made to avoid situations that allow a build-up of bacterial numbers. This is especially important in dairy herds that have been on a mastitis control program, resulting in a high percentage of cows with a low SCCs in their milk, which increases their susceptibility to coliform mastitis. The overall level of sanitation and hygiene must be improved and maintained in these herds.
Most coliform infections in periparturient cows occur very early in the dry period or just before calving, and so efforts to prevent infection should be centered on these periods. Management of the dry cow environment may provide the best opportunity for prevention of infection. While no reliable recommendations are available, cows that are housed during part or all of the day or night should be bedded on clean and dry bedding and not overcrowded, to prevent heavy fecal contamination. When possible, dry and preparturient cows are best maintained on pasture. There is an urgent need for the determination of optimum space and bedding requirements for the lounging areas of dairy cows kept under loose housing. Bedding should be kept as dry as possible. Excessively wet bedding should be removed from the back one-third of the stalls daily and replaced with fresh bedding. The addition of lime may decrease bacterial growth. Sawdust and shavings harbor more coliform bacteria than straw, and require special attention. The buildup of high numbers of coliform bacteria in the bedding of cow cubicles can be controlled by the daily removal of the sawdust from the rear of the cubicle and rebedding with clean sawdust, which is usually of low coliform count. The use of a paraformaldehyde spray on sawdust bedding reduced the coliform count for 2–3 days but it returned to its predisinfection level in 7 days. When outbreaks of coliform mastitis are encountered that are possibly associated with heavily contaminated sawdust or shavings, the bedding should be removed immediately and replaced with clean, fresh, dry straw. The use of sawdust or shavings as bedding should be avoided if possible. Sand is now considered to be the ‘gold standard’ and the most suitable alternative.
This is necessary to minimize contamination of teats. In free-stall and loose-housing dairy barns, every management technique available must be used to insure that cows do not defecate in their stalls and increase the level of contamination. This requires daily raking of the bedding in free-stall barns and adjusting head rails to insure that cows do not lie too far forward in the stall and to insure that they defecate in the alleyway.
In dairy herds that are confined for all or part of the year, the level of contamination usually increases as herd size increases; commonly the ventilation is inadequate. This leads to excessively humid conditions, which promote the development of coliform bacteria in wet bedding. This will require increased attention to sanitation and hygiene.
Postmilking teat dipping with a disinfectant has little effect on reducing the incidence of coliform mastitis because contamination of the teats occurs between milkings rather than at milking. Thus, one logical approach to the control of coliform mastitis is to reduce environmental contamination. In the event of gross fecal contamination of the udder and teats, additional time and care will be required at milking time. Premilking udder preparation can significantly influence milk quality. Lowest bacterial counts in milk are observed when the teats of cows are cleaned with water followed by thorough drying with paper towels, or when a teat disinfectant is applied to the teats followed by drying with paper towels. In addition, premilking teat disinfection in association with good udder preparation reduces the rate of intramammary infections by environmental pathogens by about 51% compared with good udder preparation only.62
Many dairy producers have now incorporated premilking teat disinfection into their mastitis control strategy, and many different teat dips are being used.62 Premilking teat dips containing 0.25% iodine, 0.1% iodophor, 0.25% iodophor and 0.55% iodophor–1.9% linear-dodecyl benzene sulfonic acid have been evaluated and have provided consistent results. Premilking and postmilking teat disinfection, in association with good udder preparation, are significantly more effective in prevention of environmental pathogen intramammary infection than good udder preparation and postmilking teat disinfection. No chapping or irritation of teats was observed. However, premilking teat disinfection did not reduce the incidence of clinical mastitis.
A latex barrier teat dip that formed a physical seal between the teat and the environment reduced the incidence of new coliform intramammary infections during lactation. The efficacy of this barrier product was thought to be due to the persistency of the dip on teats between milkings; however, it was not consistently successful. A barrier teat dip containing 0.55% chlorhexidine was effective in reducing intramammary infection associated with both environmental and contagious pathogens. The incidence of E. coli intramammary infections was reduced but the incidence of Serratia spp. and Pseudomonas spp. was increased, while the incidence of environmental streptococcal intramammary infection was unchanged by using the experimental barrier dip compared with the results using a 1% iodophor dip.
Vitamin E or selenium deficiency decreases neutrophil chemotaxis into the mammary gland and decreases the intracellular killing of bacteria by neutrophils. It is therefore important to ensure that vitamin E and selenium intakes are adequate; this is best achieved by daily ingestion of 1000 IU vitamin E and 3 mg selenium for dry cows and daily ingestion of 400–600 IU vitamin E and 6 mg selenium for lactating cows.
Considerable movement of coliform bacteria can occur from the teat apex into the teat sinus in cows that are not being milked, and so cows that are due to calve should be kept on grass or moved into a clean area at least 2 weeks before calving, their udders and teats washed daily if necessary, and teat dipping with a teat disinfectant begun 10 days before calving. This is particularly necessary for older cows and those that are known to be easy milkers. The teats of those cows that are ‘leakers’ just before calving may have to be sealed with adhesive tape or collodion to minimize the chance of infection.
Cows that are recumbent and unable to stand (e.g. the downer cow) should be well bedded on clean dry straw; their udders should be kept clean and dry, and the teats should be dipped with a teat disinfectant. Strict hygiene must be practiced when using teat siphons and teat creams, and strict asepsis observed when doing teat surgery.
Irregular vacuum fluctuations in the milking machine may induce coliform mastitis in quarters exposed to a high level of contamination. The operation and sanitation of the milking machine, especially those parts in direct contact with the teats, must therefore be examined.
The vaccination of cows during the dry period and early lactation with core lipopolysaccharide antigen vaccine (such as the Re mutant Salmonella typhimurium or the Rc mutant E. coli O111:B4 (J5 vaccine)) provides one tool to reduce the incidence and severity of clinical coliform mastitis.63 These vaccines are available in the USA and are based on mutated Gram-negative bacteria with exposed core antigens of lipopolysaccharide. The core antigen (lipid A component) is uniform between bacterial species possessing lipopolysaccharide and is immunogenic. On theoretical grounds, the Re mutant (S. typhimurium) should provide better protection than the Rc mutant (E. coli J5) because the lipid A component is more accessible to the immune system; however, comparative studies of vaccine efficacy have not been performed.
The Re and Rc mutant vaccines are protective against natural challenge to Gram-negative bacteria, and in most, but not all studies, reduce the incidence and severity of clinical Gram-negative bacterial mastitis in lactating dairy cows. In a prospective cohort study in two commercial dairy herds, during the first 90 days of lactation, cows vaccinated with E. coli J5 vaccine were at five times lower risk of developing clinical coliform mastitis than unvaccinated cows. This is corroborated with the observation that cows with naturally occurring serum IgG ELISA titers higher than 1:240 against the Gram-negative core antigen of E. coli J5 had 5.3 times lower risk of developing clinical coliform mastitis than cows with lower titers. Vaccination reduced the severity of clinical signs following intramammary experimental challenge with a heterologous E. coli strain.64 In cows vaccinated with the J5 bacterin at drying off, at 30 days after drying off and within 48 hours after calving, and challenged 30 days after calving with a strain of E. coli known to cause mild clinical mastitis, the duration of intramammary infection and local signs of mastitis were reduced compared to controls.63 Also, the concentrations of bovine serum albumin in milk 24 hours after challenge were greater in control cows than in vaccinated cows.
A partial budget analysis of vaccinating dairy cattle with one core lipopolysaccharide antigen vaccine (the Rc mutant of E. coli or J5 strain) indicated that herd vaccination programs were predicted to be profitable when more than 1% of cow lactations resulted in clinical coliform mastitis65 and predicted to be profitable at all herd milk production levels.
Core lipopolysaccharide antigen vaccines have the potential to have deleterious effects because of their endotoxin content. For instance, vaccination of late lactation and dry cattle with the S. typhimurium Re mutant transiently decreased leukocyte and blood segmented neutrophil concentration, but the decrease is probably clinically insignificant.66 This response is typical for endotoxin administration. Vaccination of lactating dairy cattle with the E. coli Rc mutant decreased milk production by 7% at the second and third milkings after vaccination.67 These two studies indicate that core lipopolysaccharide antigen vaccines should not be administered to diseased cattle or to healthy cattle in hot and humid weather, because of their decrease in cardiovascular reserve. The vaccines should not be administered at the same time as other Gram-negative vaccines.
Hogan J, Smith KL. Coliform mastitis. Vet Res. 2003;34:507-519.
Wilson DJ, Gonzalez RN. Vaccination strategies for reducing clinical severity of coliform mastitis. Vet Clin North Am Food Anim Pract. 2003;19:187-197.
Smith GW. Supportive therapy of the toxic cow. Vet Clin North Am Food Anim Pract. 2005;21:595-614.
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3 Shuster DE, et al. J Dairy Sci. 1991;74:3407.
4 Hogan JS, et al. J Dairy Sci. 1995;78:2502.
5 Erskine RJ, et al. J Am Vet Med Assoc. 1991;198:980.
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7 Jones TO. Vet Bull. 1990;60:205.
8 Green MJ, et al. Vet Rec. 1996;138:305.
9 Shuster DE, et al. Am J Vet Res. 1996;57:1569.
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11 Kremer WDJ, et al. J Dairy Sci. 1993;76:3428.
12 Paape MJ, et al. Am J Vet Res. 1996;57:477.
13 Weiss WP, et al. J Dairy Sci. 1997;80:1728.
14 Hogan JS, et al. J Dairy Sci. 1992;65:399.
15 Maddox JF, et al. Prostaglandins. 1991;42:369.
16 Todhunter DA, et al. Am J Vet Res. 1991;52:184.
17 Vandeputte-Van Messom G, et al. J Dairy Res. 1993;60:19.
18 Tyler JW, et al. North Am Food Anim Pract Vet Clin. 1993;9:537.
19 Jones GF, Ward GE. J Am Vet Med Assoc. 1990;197:731.
20 Lohuis JACM, et al. J Dairy Sci. 1990;73:333. 342
21 Hill AW. Proceedings of the 30th Annual Meeting of the National Mastitis Council. Verona, WI: National Mastitis Council, 1991;6.
22 Welles EG, et al. Am J Vet Res. 1993;54:1230.
23 Shuster DE, et al. Am J Vet Res. 1993;54:80.
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25 Cebra CK, et al. J Vet Intern Med. 1996;10:252.
26 Wenz JR, et al. J Am Vet Med Assoc. 2001;219:976.
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45 Erskine RJ, et al. Am J Vet Res. 1995;56:481.
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47 Kutila T, et al. J Vet Pharmacol Ther. 2004;27:197.
48 Dosogne H, et al. Inflamm Res. 2002;51:201.
49 Monfardi E, et al. Vet Immunol Immunopathol. 1999;67:373.
50 Shpigel NY, et al. Vet Rec. 1998;142:135.
51 Bezek DM. J Am Vet Med Assoc. 1998;212:404.
52 Guterbock WM, et al. J Dairy Sci. 1993;76:3437.
53 Van Enennaam AL, et al. J Dairy Sci. 1995;78:2086.
54 Goldberg JJ, et al. Am J Vet Res. 1995;56:440.
55 Tyler JW, et al. J Am Vet Med Assoc. 1994;204:1949.
56 Tyler JW, et al. Am J Vet Res. 1994;55:278.
57 Roeder BL, et al. Am J Vet Res. 1997;58:549.
58 Shpigel NY, et al. Res Vet Sci. 1994;56:62.
59 Shpigel NY, et al. J Vet Med A. 1996;43:331.
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61 Green MJ, et al. Vet Rec. 1997;140:149.
62 Oliver SP, et al. J Food Prot. 1993;56:852.
63 Hogan JS, et al. J Dairy Sci. 1995;78:285.
64 Hogan JS, et al. J Dairy Sci. 1992;75:415.
65 DeGraves FJ, Fetrow J. J Am Vet Med Assoc. 1991;199:451.
66 Scott HM, et al. J Dairy Sci. 1998;81:1928.
67 Musser JMB, Anderson KL. J Am Vet Med Assoc. 1996;209:1291.
Etiology Streptococcus uberis, Streptococcus dysgalactiae spp. dysgalactiae, other Streptococcus spp. most commonly; occasionally Enterococcus spp.
Epidemiology Common cause of subclinical and clinical mastitis in herds and countries that have controlled contagious mastitis. Responsible for approximately one-third of all cases of clinical mastitis in herds without contagious pathogens. Rate of infection high during first 2 weeks following drying off and 2 weeks before calving. Duration of infection usually short (<8 d). Prevalence of infection at calving: 11% of cows and 3% of quarters. Bedding materials (high in straw bedding) most important source of environmental streptococci; bacteria can be isolated from many different feedstuffs and several locations on cow (teats, rumen, feces, saliva, lips, nares). Bacterial numbers low in sand, which is bedding of choice
Clinical findings Abnormal milk, abnormal gland, usually no systemic signs. Recovery in two to three milkings
Clinical pathology Culture of milk
Necropsy findings Not applicable
Diagnostic confirmation Culture bacteria from milk and milk SCC
Differential diagnosis Cannot differentiate from other causes of subacute and acute mastitis without culture of milk
Treatment Antimicrobial intramammary infusions increase bacteriological cure rate and decrease percentage of relapses. Intramammary antibiotics should be administered to all clinical cases of mastitis due to environmental streptococci
Control Decrease exposure of teat end to pathogens by attention to environment, dry bedding, sand for bedding, premilking hygiene and premilking germicide teat dipping. Dry cow therapy with penicillin G, cloxacillin, erythromycin and first-generation (cephapirin) or third-generation (ceftiofur) cephalosporins. Application of an internal teat sealant of bismuth subnitrate at dry-off may decrease new infection rate in dry period
Streptococcus uberis and Streptococcus dysgalactiae spp. dysgalactiae and the enterococci are the most commonly isolated environmental streptococci from intramammary infections. Other uncommon environmental streptococci involved in bovine mastitis include Streptococcus equi var. zooepidemicus,1 Streptococcus viridans, S. equinus (S. bovis), Streptococcus spp. group G, Streptococcus pyogenes and Streptococcus pneumoniae. Both S. uberis and S. dysgalactiae are widespread in the animal’s environment and on the skin of the teats. Two genotypes of S. uberis (S. uberis = type 1; Streptococcus paruberis = type II), with marked differences in their capacity to invade quarters and to cause mastitis, have been identified.2,3 Enterococcus spp. are also a common cause of environmental intramammary infections.4 The streptococcal species most commonly isolated from intramammary infections all hydrolyze esculin (S. uberis, Streptococcus spp., Enterococcus spp.).
In countries where the prevalence of intramammary infections due to the contagious pathogens S. agalactiae and S. aureus has been reduced or eradicated, the proportion of intramammary infections associated with environmental streptococci has increased markedly; these organisms are a leading cause of both subclinical and clinical mastitis in dairy cattle worldwide.4,5 S. uberis is now a common cause of intramammary infection occurring during the dry period, with most clinical cases occurring during the first part of lactation. Many infections acquired during the dry period persist to lactation and contribute to the incidence of clinical mastitis in early lactation. The rate of new infection due to environmental streptococci is elevated during the first 2 weeks following drying off and the 2 weeks prior to calving; the rate of new infections is greater during the first month of lactation than during the remainder of the lactation. Approximately 50% of new infections occur during the dry period and 50% in the early part of lactation. The rate of new infections during the dry period is about five times greater than during lactation.4 Based on data from surveys of milk samples over a 10-year period, the point prevalence of infection of environmental streptococci was 4% of quarters and 12% of cows.6 The percentage in heifers at calving is similar to that in cows. The prevalence of environmental streptococci isolation at drying off and calving was 2.5% and 3.0%.6 Environmental streptococcal intramammary infections are usually short-lived (< 28 days), with only a small percentage becoming chronic.4,7
The most important change in the epidemiology of bovine mastitis over the past decade has been the rise in the importance of environmental pathogens, mainly causing clinical mastitis, relative to contagious pathogens. Remarkable increases in both the coliforms and environmental streptococci as causes of clinical mastitis have occurred.5 The percentage of clinical cases of mastitis from which environmental streptococci can be isolated ranges from 14% in Ontario to 26% in the UK. When expressed as a percentage of clinical cases from which a major pathogen was isolated, environmental streptococci are isolated in 37–45% of cases.
The environmental streptococci, especially S. uberis, have been isolated from bedding materials and the lips and tonsils of cows, with the abdominal skin of cows often harboring the largest population.8 Some cows are permanently colonized with S. uberis and may pass large numbers of the bacteria in the feces. Fecal shedding is believed to play an important role in the maintenance of S. uberis populations on dairy farms, and is the likely source of large numbers of the organism in straw bedding on farms where this form of mastitis persists.9,10 The numbers of environmental streptococci in organic bedding materials vary with the type of bedding. Large numbers of S. uberis are found in straw bedding and much lower numbers in sawdust and wood shavings. The numbers of streptococci recovered from the teats of cows bedded on sawdust are lower than those bedded on straw. Long straw used in calving box stalls or as bedding in loose housing can be a source of considerable exposure to environmental streptococci.
S. dysgalactiae can also be found in the environment of dairy cattle and has been isolated from the tonsils, mouth and vagina, and the mammary glands. It has characteristics of both a contagious and an environmental pathogen and some categorization schemes place it in the contagious category, although it is primarily an environmental pathogen. S. dysgalactiae is also associated with summer mastitis, which affects dry cows and heifers during the summer months. It has been isolated from the common cattle fly Hydrotaea irritans, which may be involved in the establishment and maintenance of bacterial contamination of teats. S. dysgalactiae may colonize the teat prior to infection with A. pyogenes and anaerobic bacteria such as P. indolicus and F. necrophorum.
The major risk factor for environmental streptococci infection is exposure of the teat end to mastitis pathogens in the environment. Transmission is predominantly from the environment.7 Exposure of uninfected teats to environmental streptococci can occur during the milking process, between milkings, during the dry period and prior to parturition in first-lactation heifers. The rate of new infections is greatest during the summer months in North America.4
Housing and management practices on dairy farms may contribute to contamination of bedding materials and exposure of teats to environmental streptococci. Housing facilities that predispose to the accumulation of feces on cows will increase the rate of exposure of the teat end to the pathogens. Straw bedding appears to increase the risk of S. uberis mastitis,11 and an increase in S. uberis mastitis cases occurs when cows are housed in deep straw pack.
Pastured cattle are generally at reduced risk for environmental streptococcal mastitis when compared to cows in confinement housing. However, certain pasture conditions, such as areas under shade trees, poorly drained ground surfaces, ponds and muddy areas, may result in a high rate of exposure to the pathogens. The environmental streptococci are the most significant environmental pathogen in New Zealand dairy herds where cows spend almost 100% of their time on pasture.12
S. dysgalactiae is commonly isolated from heifers and cows in the dry period and is one of the most prevalent pathogens isolated from cases of summer mastitis. The spread of S. dysgalactiae between cows within dairy herds may occur directly or by way of the milking machine or environment.
The risk of new infections is influenced by the stage of lactation and parity of the cow. The rate of new infection is highest during the 2 weeks following drying off and the 2 weeks prior to calving. The high rates of new infection following drying off may be associated with the lack of flushing action of milking, changes in the composition of the mammary secretion, which may enhance the growth of the pathogens, and the lack of a keratin plug in the streak canal. The primary defense mechanisms for S. uberis are the length of the teat canal and the amount of keratin in the lining.13 Antimicrobial dry cow therapy reduces the infection rate in the early part of the dry period but has no or little effect on preventing infection with S. uberis at the end of the dry period. The increase in susceptibility to infection just prior to parturition may be associated with the lack of milking when the gland is accumulating fluid, loss of keratin plugs from streak canals, or immunosuppression of the periparturient period. The rate of infection is also higher in older cows than for either heifers or cows in second lactation, and highest during the summer months for both cows in lactation and cows in the dry period. This is in contrast to contagious pathogens, where exposure occurs primarily during the milking process.
S. uberis is ubiquitous in the cow’s environment with multiple environmental habitats.3 Consequently the mammary gland is exposed continuously to the pathogen during lactation and the dry period and infections are associated with a large variety of strains. Several virulence factors of S. uberis and S. dysgalactiae have been identified that are important in the pathogenesis of environmental mastitis. Antiphagocytic factors allow S. uberis to infect and multiply in the gland, and to adhere to and invade the mammary tissue.14 Bovine mammary macrophages are capable of phagocytosis of the organism but certain strains of S. uberis are capable of resisting phagocytosis by neutrophils, because of their hyaluronic acid capsule.15,16 The ability of S. uberis to invade the bovine mammary epithelial cells could result in chronic infection and protection from host defense mechanisms and the action of most antimicrobial agents,17 which may explain the intractable response to therapy in some cases. However, most ‘intractable’ infections are due to an inappropriately short duration of treatment.
S. dysgalactiae behaves like both a contagious and an environmental pathogen and can invade bovine mammary epithelial cells, which may explain the persistence of infection.18 Different biotypes of S. dysgalactiae have been identified,19 and strains can possess several antiphagocytic factors, including M-like protein, alpha-2-macroglobulin, capsule and fibronectin binding, and virulence factors, including hyaluronidase and fibrinolysin.
An existing intramammary infection due to C. bovis is a risk factor for environmental streptococcal infection20 through an unidentified mechanism.
The major economic losses associated with environmental streptococcal mastitis are caused by clinical mastitis resulting in lost production, milk withholding, premature culling, increased labor, and costs of therapy and veterinary services. 88% of the loss associated with clinical mastitis is attributed to loss of milk production and milk withholding. Pluriparous cows lost 2.6 times as much as first-calf heifers, and cows less than 150 days in milk lost 1.4 times more than cows more than 150 days in milk.
Infections with S. dysgalactiae artificially induced in goats are indistinguishable from mastitis associated with S. agalactiae and the pathogenesis is probably similar in all streptococcal mastitides.
In experimental infection of dairy cows with S. uberis there is acute inflammation, resulting in the accumulation of large numbers of neutrophils in the secretory acini in 24 hours.21 After 6 days, the neutrophil response is still evident but there is cellular infiltration, septal edema, extensive vacuolation of secretory cells, focal necrosis of alveoli, small outgrowths of the secretory and ductular epithelium, and widespread hypertrophy of the ductular epithelium. The organism is present free or phagocytosed, in macrophages in the alveolar lumina, adherent to damaged secretory or ductular epithelium, in the subepithelium and septal tissue, and in lymphatic vessels and lymph nodes. The macrophage is important as the primary phagocytic cell but the marked neutrophil response may be ineffective as a defense mechanism. It is hypothesized that the marked neutrophil response following infection with S. uberis, rather than the organism, may be responsible for the most of the effects of the mastitis.21 The current consensus is that environmental streptococci (with the possible exception of S. dysgalactiae) are not contagious pathogens.
Approximately 50% of environmental streptococcal intramammary infections cause clinical mastitis during lactation. Clinical abnormalities occur in 42–68% of these infections in the same herd in different years.4 The clinical findings are usually limited to abnormal milk or abnormal gland. In about 43% of cases the findings are limited to abnormal milk, 49% involve abnormal milk and an enlarged (abnormal) gland, and in only 8% of cases are there systemic signs with a fever and anorexia (abnormal cow). Clinical recovery commonly occurs in 24–48 hours.
The laboratory diagnostic tests for these pathogens are the same as for S. agalactiae. All the environmental streptococci except S. dysgalactiae hydrolyze esculin on blood agar. Species can be differentiated with reasonable success using a variety of biochemical tests, such as the API20 Strep and serological grouping using specific antisera of Lancefield groups.3 However, molecular biological techniques are needed for detailed epidemiological studies.16 The HYMAST® diagnostic kit is available as a field test to distinguish between Gram-positive and Gram-negative bacteria in the milk of dairy cows with clinical mastitis.5
The in vitro susceptibility of environmental streptococci to antimicrobial agents is high. Most isolates of S. uberis and S. dysgalactiae are susceptible to penicillin, novobiocin, amoxicillin and cephapirin. A high percentage (96%) are also susceptible to tetracycline, but susceptibility to aminoglycosides is much lower. Most cases of clinical mastitis associated with S. uberis and S. dysgalactiae respond well to intramammary infusions of penicillin, cephalosporins, cloxacillin, erythromycin and tetracyclines. Spontaneous cures can also occur. Clinical cases in lactating cows should be treated by at least two intramammary infusions 12 hours apart; this may produce clinical cure but fail to produce a bacteriological cure. Subclinical infections in late lactation may be left until the dry period. For clinical cases in the first 100 days of lactation there is substantial economic benefit from treatment. Some cases associated with strains of S. uberis appear intractable to treatment; extended treatment is necessary in these animals. Failure of treatment may be due to epithelial cell invasion and movement of the bacteria into subepithelial layers, possibly reducing the effectiveness of the antimicrobial. Extended therapy (for 5 or 8 days) with intramammary ceftiofur (125 mg), pirlimycin (50 mg) or penethemate hydriodide, dihydrostreptomycin sulfate and framycetin sulfate, every 24 hours, increases the bacteriological cure rate for cattle with experimentally induced S. uberis mastitis.10,22-24 In a study of 1148 cases of subclinical environmental streptococci mastitis in New York, commercially available intramammary infusions were more effective than untreated controls (66% bacteriological cure), with the following bacteriological cure rates: amoxicillin (90%), penicillin (82%) and cloxacillin (79%).25
Treatment using oxytocin and frequent stripping of the affected glands without intramammary antibiotic administration is not recommended because cure rates are much lower.23,26 Moreover, not administering antimicrobial agents results in a higher relapse rate.27,28 Many of the relapses were associated with the environmental streptococci and therefore intramammary antimicrobial treatment should be routinely performed. In particular, because clinical mastitis with an abnormal gland or abnormal cow induces some pain and discomfort in the cow, withholding an effective treatment (antimicrobial agents) cannot be condoned on animal welfare grounds.
Reinfection may occur quickly if the contributory causes are not corrected. Infections with S. zooepidemicus do not respond well to treatment with penicillin. Mastitis associated with S. pneumoniae responds well to local treatment with penicillin in large doses (300 000 units per infusion) but quarters allowed to go without treatment for any length of time suffer complete loss of function. All cases of mastitis associated with this bacteria should receive parenteral treatment with penicillin.
The control of mastitis due to environmental streptococci is achieved by decreasing the exposure of pathogens to the teat end and by increasing the resistance to intramammary infections. A specific control recommendation for environmental streptococci mastitis is not to bed on straw, but this may not be a practical or economic recommendation for some producers. If straw bedding is used, a reduction in the teat end exposure to S. uberis can result from frequent (daily) replacement of bedding.
Reducing the exposure of the teat end to pathogens depends on maintenance of a clean and dry environment. Special attention must be directed to the dry cow and close-up heifer housing, the calving area, lactating cow housing, and the milking parlor and milking hygiene. Organic bedding materials such as straw that support large numbers of environmental pathogens should be kept dry. Sand is the ideal bedding material because it has the lowest number of coliform and environmental pathogens. Wet and damp areas in the back part of free stalls and tie stalls promote exposure to environmental pathogens. Milking time hygiene should emphasize milking of clean, dry teats and udder, with a properly functioning milking machine. Predipping with a teat dip germicide may reduce environmental mastitis by as much as 50% but this reduction does not occur in all herds.
Dry cow therapy to prevent new infections has not been as successful for the control of all causes of environmental mastitis as it has been for contagious mastitis. However, dry cow therapy is more effective against the environmental streptococci than against coliform bacteria.29 Application of an internal teat sealant of bismuth subnitrate at dry-off is effective in preventing infections associated with S. uberis during the dry period.30,31
A long-acting intramammary infusion dry cow therapy containing 250 mg cephalonium administered after the last milking of lactation reduced the incidence of new infections due to S. uberis from 12.3% to 1.2%.32 Clinical infections during the dry period were most prevalent in quarters identified as having open teat canals. Fewer open teat canals were observed among treated quarters over the first 4 weeks of the dry period. It is proposed that the teat canal of treated quarters closed earlier than those of untreated quarters. Most of the new infections in the untreated controls occurred within the first 21 days of the dry period. Normally, the teat canal is dilated for up to 7 days after drying off, with a keratin plug then forming over the following 14–21 days. It is suggested that once a physical keratin seal has formed in the teat canal after drying off, an uninfected quarter has a very low risk of infection over the remainder of the dry period. Treated quarters had a lower incidence of new clinical infections during the next lactation and lower SCCs.
Experimentally, multiple intramammary vaccinations with whole killed S. uberis cells resulted in complete protection against experimental infection in cattle.33 Bacteria could not be isolated from the quarters after challenge, and protection occurred in the absence of a marked neutrophil response. Preparations containing plasminogen activator may form the basis of a vaccine against S. uberis.34 Vaccines are presently commercially unavailable,16 and vaccination is not currently recommended as part of the control program for mastitis due to environmental streptococci.
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