Contagious pathogens

Environmental pathogens

Environmental Streptococcus species include S. uberis and S. dysgalactiae, which are the most prevalent; less prevalent is S. equinus (formerly referred to as S. bovis). The environmental coliforms include the Gram-negative bacteria E. coli, Klebsiella spp. and Enterobacter spp. A. pyogenes mastitis can be an important problem in some herds.

Prevalence

In most countries, surveys in dairy herds indicate that the prevalence of infection of mastitis pathogens is approximately 50% of cows and 10–25% of quarters. The prevalence of infection in dairy heifers of breeding age and in pregnant dairy heifers varies widely¹ from 30–50% of heifers and 18% of quarters² to as high as 97% of heifers and 75% of quarters.³

Incidence

The average annual incidence of clinical mastitis, calculated as the number of clinical quarter cases per 100 cows at risk per year, including the dry period, in individual herds ranges from 10–12% in most herds⁴ but higher incidences, ranging from 16–65%, occur in some herds.^5,6 The greatest risk of first acquiring mastitis occurs early in lactation, usually in the first 50 days.⁷ The risk of clinical mastitis also increases with increasing parity.⁷ In beef herds, 32–37% of cows and 18% of quarters may have intramammary infection, which has a significant negative effect on calf weaning weights.⁸

Case fatality rates vary widely depending largely on the identity of the causative organism. For example, S. agalactiae mastitis is not a fatal disease but peracute staphylococcal mastitis in a recently calved cow often may be fatal. Details of the occurrence of the different types of mastitis are presented in their individual sections in this chapter.

Contagious pathogens

The prevalence of infection with S. aureus in cows ranges widely, usually from 7–40%, but higher in some herds.⁹ A survey of Danish dairy herds found that 21–70% of all cows and 6–35% of all quarters were infected.¹⁰ S. aureus was isolated from 10% of quarter samples and was the most common species isolated.¹⁰ The prevalence of streptococci, including S. agalactiae, ranges from 1–8% of cows. A relative incidence of S. agalactiae, other streptococci and S. aureus of 1:1:2 is a common finding. S. aureus may still assume some importance as a cause of subclinical mastitis but its prevalence has been reduced by modern mastitis control programs, leading to a higher proportion of culture-negative mastitic quarters and a corresponding, and perhaps consequent, increase in infections by E. coli and Klebsiella spp. The prevalence of infection due to Mycoplasma spp. varies widely.⁹

The prevalence of infection due to an individual pathogen, and therefore the ratio between its incidence and that of other pathogens, depends on several risk factors such as size of herd and quality of management, especially milking parlor hygiene and cleanliness of accommodation, and parity of animal (heifer or cow). For example, large, zero-grazed herds kept in drylot conditions are likely to encounter more hygiene problems than conventionally housed herds mainly because of constant soiling of the udder by inadequate or improper bedding in larger units. In those circumstances there is likely to be a much higher prevalence than usual of mastitis associated with E. coli and S. uberis.

Teat skin opportunistic pathogens

Coagulase-negative staphylococcal species were found in 4.1% of samples; the most frequently isolated were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%).

Environmental pathogens

The prevalence of intramammary coliform infections in a dairy herd seldom exceeds 1–2%; the prevalence of intramammary environmental streptococci is less than 5% in well managed herds but may exceed 10% in some problem herds.¹¹ A characteristic of intramammary coliform infections is the short duration: 40–50% persist less than 7 days. The majority of environmental streptococci infections last less than 30 days. In a survey of Danish dairy herds, S. dysgalactiae (1.6%) and S. uberis (1.4%) were the second and third most common species isolated.

Heifers

Surveys of intramammary infection of heifers in regions such as Louisiana indicate variability in prevalence and duration of intramammary infection according to species of bacteria present around the time of parturition. About 20% of heifers were infected with S. aureus and 70% with coagulase-negative staphylococci, the minor pathogens that are part of the normal teat skin flora of heifers.¹² S. chromogenes was isolated from 15% of all quarters of heifers before parturition but decreased shortly after parturition to 1%.¹³ Up to 97% of breeding age and pregnant dairy heifers and 75% of their quarters may be infected with S. aureus, S. hyicus and S. chromogenes.³ Infections with S. simulans and S. epidermidis occurred in 1–3% of quarters both before and after parturition. S. dysgalactiae was isolated from 4–6% of quarters before and immediately after parturition. Intramammary infections with S. aureus rarely occurred before parturition but increased during the first week after parturition. There was no association between the prevalence of S. aureus in heifers before parturition and the prevalence in lactating cows.

Contagious pathogens

S. agalactiae and S. aureus reside primarily in the udder of infected cows; the source of infection is other infected cows and exposure to uninfected quarters is limited to the milking process.

Teat skin opportunistic pathogens

A number of species of coagulase-negative staphylococcus reside primarily on the teat skin of cattle.

Environmental pathogens

S. uberis, S. dysgalactiae, and coliforms are common inhabitants of the cow’s environment such as bedding. The exposure of uninfected quarters to environmental pathogens can occur at any time during the life of the cow, including milking time, between milkings, during the dry period and prior to first calving in heifers.

Animal risk factors

Environmental and management risk factors

Pathogen risk factors

Subclinical mastitis

Total milk losses from quarters affected with subclinical mastitis have been estimated to range from 10–26%.³⁷ Lower SCCs are associated with higher milk production, and rolling herd average milk production has been estimated to decrease by 190 kg per unit increase of linear somatic cell score. Most estimates indicate that on average an affected quarter results in a 30% reduction in productivity, and an affected cow is estimated to lose 15% of its production for the lactation. This loss is sometimes expressed as a loss of about 340 kg of saleable milk, due to loss of production and the value of milk that has to be withheld from sale. The loss in production by an infected quarter may be largely compensated by increased production in the other quarters so that the net loss from the cow may be less than expected. In addition to these losses, there is an added loss of about 1% of total solids by changes in composition (fat, casein, and lactose are reduced and glycogen, whey proteins, pH, and chlorides are increased), which interferes with manufacturing processes, and other losses include increased culling rates and costs of treatment. Comparisons between low- and high-prevalence herds always show a financial advantage of about 20% to the low-prevalence herds, the gain varying with the local price of milk or butter fat. In beef herds the losses are in the form of rare deaths of cows and failure of the calves to gain weight.

Approximately 75% of the economic loss from subclinical mastitis is attributable to loss of milk production. Other costs include discarding milk from treated cows, drug costs, veterinary costs, labor and loss of genetic potential of culled cows.

Clinical mastitis

Clinical mastitis results in marked decreases in milk production, which are much larger in early than late lactation. Milk production losses are also greater in cows with multiple lactations than first-lactation cows, and clinical mastitis also decreases the duration of lactation and increases the likelihood of culling. Clinical cases of brief duration that occur after the peak of lactation affect milk production very little but can induce abortion during the first 45 days of gestation.³⁶ Clinically affected quarters may not completely recover milk production in subsequent lactations but these carry-over losses are not as large as the losses from acute mastitis. In the National Animal Health Monitoring System of dairy herds in the US, clinical mastitis alone was the most costly disease identified, at a loss to the producer of $27–50 per cow per year.³⁷

The costs of clinical mastitis and mastitis prevention in dairy herds have been estimated, based on monitoring 50 dairy herds over 1 year.³⁸ Mean incidence of clinical mastitis was 39 cases/100 cow-years; each clinical case cost $38/cow-year, with a mean cost per clinical episode of US$107. Prevention of mastitis cost $14.50/cow-year.³⁹ Lost milk production was estimated at $14.85/cow-year, which does include the losses associated with subclinical mastitis.

The component causes of economic loss associated with mastitis outlined above vary according to the causative pathogen and are described under specific mastitides. In general terms S. aureus and E. coli may cause death from peracute mastitis; A. pyogenes causes complete loss of quarters; staphylococci and streptococci cause acute clinical mastitis, but their principal role is in causing subclinical mastitis, resulting in a reduction of milk produced and a downgrading of its quality. Of these, S. agalactiae causes the greatest production loss, whereas S. aureus has the higher infection rate, greater resistance to treatment and longer duration of infection. At one time S. aureus represented the impassable barrier to mastitis control programs.

Other factors that affect the magnitude of the loss associated with mastitis include age (the loss is greatest in mature cows), and when the attack occurs in the first 150 days of lactation.

Bulk tank milk somatic cell counts

The SCC of bulk tank milk is an indirect measure of the prevalence of mastitis within a dairy herd. The SCC is increased primarily, but not exclusively, because of subclinical mastitis associated with Gram-positive bacterial intramammary infections. There is a good correlation between the number of streptococci (S. agalactiae, S. dysgalactiae, and S. uberis) colony-forming units found in bulk tank milk and its SCC. The number of colony forming units (cfu) of S. aureus is moderately correlated to the bulk tank milk SCC.⁴⁶ As contagious mastitis has become more effectively controlled, environmental mastitis pathogens have become a relatively more important cause of high SCC in bulk tank milk, especially in herds with moderate (<400000 cells/mL) to low (<150000 cells/mL) bulk tank milk SCC.

The association between management practices, dairy herd characteristics and SCC of bulk tank milk has been examined in about 60000 cows in 843 herds over a 2-year period.⁴⁷ Results indicated that the prevalence of S. agalactiae and S. aureus intramammary infections was associated with bulk tank SCC.⁴⁷ In herds free of S. agalactiae mastitis, the prevalence of S. aureus and C. bovis intramammary infections were correlated with bulk tank SCC. For herds without S. agalactiae, use of sawdust bedding was associated with a decrease in SCC in bulk tank milk, while a dirty loose housing area was associated with an increase in SCC in bulk tank milk. Increased milk production, repeated mastitis control visits and use of particular predip compounds were significantly associated with decreased SCC in bulk tank milk in all herds, regardless of whether any cows in the herd had S. agalactiae mastitis. In herds with S. agalactiae mastitis, use of iodine, chlorhexidine, peroxide or sodium chlorite–lactic acid as a predip was associated with a decrease in SCC of bulk tank milk.⁴⁷

The SCC of bulk tank milk has become a widely used test because it provides a sensitive and specific indicator of udder health and milk quality. The sample for analysis is obtained by agitating the milk for 5–10 minutes and collecting a sample from the top of the bulk tank milk using a clean dipper. The sample should not be collected near the outlet valve because this varies from that in the rest of the tank. The SCC of bulk tank milk is widely used to regulate whether milk may be legally sold and to determine the price paid for raw milk. Premium and penalty payments are calculated on the basis of 3-month geometric mean of weekly bulk milk tank SCC measurements. Milk processing plants in most developed countries use automatic electronic somatic cell counters routinely in order to provide a monthly report of the bulk tank milk SCC. The test requires only that the sample for examination be taken randomly and not frozen, that it be prepared with the correct reagent, that the laboratory counter be set at the right calibration and that the sample be examined quickly or preserved with formalin to prevent cell losses during storage. The bulk tank milk SCC is extremely useful in creating awareness of the existence of a mastitis problem, so that when the SCC of bulk tank milk exceeds permissible limits further investigation of the herd is indicated. In a seasonal herd in which all cows are at the same stage of lactation the bulk milk cell count will normally be high in early lactation and just before drying off. To overcome these and other factors that are likely to transiently influence bulk tank milk SCC, it is recommended that correction factors be introduced into the estimation or that a rolling SCC, in which monthly data are averaged for the preceding 3 months, be used. Consideration of this figure will avoid too hasty conclusions on one high count caused by an extraneous factor.

It is not possible to use the bulk tank milk SCC to determine the number of cows in a herd affected by mastitis but it is possible to estimate fairly accurately the number of infected quarters. In general, as the bulk tank milk SCC increases, the prevalence of infection increases and losses in production increase. Production losses calculated as a percentage of production expected with a count of 200000 cells/mL are shown in Table 15.1. A bulk tank milk SCC of more than 300000 cells/mL is considered to indicate a level of mastitis in the herd that warrants examination of individual cows. Herds with a high bulk tank milk SCC have significantly lower production levels and are less likely to use a postmilking teat dip or to have a regular program of milking machine maintenance or automatic cluster removal.⁴⁶

Table 15.1 Estimated prevalence of infection and losses in milk production associated with bulk tank milk somatic cell count

Culture of bulk tank milk

Bacteria present in bulk tank milk may originate from infected udders, from teat and udder surfaces or from a variety of other environmental sources; however, despite the large number of potential sources for bacteria, culture of bulk tank milk is a useful technique for screening for major mastitis pathogens.⁴⁸ The culture of S. aureus and S. agalactiae from bulk tank milk is a reliable indicator of infection by those pathogens in the herd. The number of those pathogens found on culture is determined by the number of bacteria shed, the number of infected cows, the milk production level of infected cows relative to herd mates, and the severity of infection. A single culture of bulk tank milk has low sensitivity but high specificity for determining the presence of S. agalactiae or S. aureus in the herd. Thus many infected herds will be called negative but few uninfected herds will be called positive. Pathogens such as Nocardia spp. and Mycoplasma spp. have also been identified by culture of bulk tank milk. In general, the sensitivity of a single bulk tank milk culture to detect the presence of intramammary infections due to S. agalactiae ranges from 21–77%, for S. aureus it ranges from 9–58% and for M. bovis it is 33%.

Environmental bacteria such as S. uberis, S. dysgalactiae, and coliforms may enter milk from intramammary infections, but also from nonspecific contamination. The presence of these organisms in bulk tank milk may relate to the general level of environmental contamination and milking hygiene in the herd. Udder infections with these environmental pathogens are predominantly of short duration and characterized by clinical disease, which makes their inadvertent introduction to the bulk tank less likely.

String sampling or milk line sampling from the positive pressure side of the milking system is the collection of milk samples from a group of cattle instead of the entire herd, as in bulk tank milk sampling. String sampling may have some merit in identifying subgroups of cattle with the highest prevalence of infection. String sampling is thought to be more sensitive in monitoring herds for contagious pathogens than bulk tank milk sampling. If a production group tests positive on a string culture, then individual composite milk cultures can be performed to identify individual animals. Information of the culture results from string sampling should assist development of control programs; however, milk samples left in the pipeline from one string can confound the culture results of subsequent strings.

Numerous bacteriological techniques have been used to isolate and identify pathogens in bulk tank milk but none has been established as the gold standard method for the culture of milk from bulk tanks. The most suitable laboratory medium for growth and classification of the pathogens from bulk tank milk needs to be assessed. Sampling strategies have included weekly and monthly samples, but it remains to be determined which strategy is optimal relative to herd size and management, disease characteristics and practicality.

Culture of bulk tank milk has been compared to bulk tank SCC, herd summaries of SCC of individual cows and herd summaries of cultures of individual cows.⁴⁸ A significant correlation was found between bulk tank SCC and the frequency of isolating S. agalactiae from monthly samples of bulk tank milk. The correlations between S. aureus from bulk tank milk and SCC are lower.

Culture of individual cow milk

Individual cow milk can be cultured as part of a herd examination for mastitis, or on individual quarter samples, or on composite samples including milk from all four quarters. An intramammary infection is defined as the presence of the same pathogen in duplicate samples collected immediately after each other, or the presence of the same pathogen in two of three consecutive cultures obtained on different sampling dates. Individual quarter samples are preferred because the costs of treatment dictate that the least possible number of quarters be treated. With this technique only affected quarters are treated; if the quarter infection rate is low the saving in treatment costs is relatively large.

Milk sampling for culture must be carried out with due attention to cleanliness since samples contaminated during collection are worthless. The technique of cleaning the teat is of considerable importance. If the teats are dirty, they must be washed and then properly dried or water will run down the teat to the teat end and infect the milk sample. The end of the teat is cleaned with a swab dipped in 70% alcohol, extruding the external sphincter by pressure to insure that dirt and wax are removed from the orifice. Brisk rubbing is advisable, especially of teats with inverted ends. The first two or three streams are rejected because their cell and bacterial counts are likely to be a reflection of the disease situation within the teat rather than within the udder as a whole. The next few streams, the premilking sample, is the approved one because of its greater accuracy. For complete accuracy a premilking and a postmilking sample are taken. If tuberculosis is suspected, the last few streams are the critical ones. Indirect and chemical tests for mastitis can be carried out as accurately on foremilk as on later milk.

If individual quarter samples are collected, screw-cap vials are most satisfactory. During collection the vial is held at an angle to the ground in order to avoid as far as possible the entrance of dust, skin scales and hair. If there is delay between the collection of samples and laboratory examinations, the specimens should be refrigerated or frozen. Freezing of milk samples appears to have variable effects on bacterial counts, depending on the bacteria. A. pyogenes and E. coli counts are decreased by freezing, coagulase-negative Staphylococcus spp. counts are increased, and Streptococcus and S. aureus counts are either unaffected or increased by about 200%.⁴⁹

The laboratory techniques used vary widely and depend to a large extent on the facilities available. Incubation on blood agar is most satisfactory, selective media for S. agalactiae having the disadvantage that other pathogens may go undetected. Smears of incubated milk are generally unsatisfactory as not all bacteria grow equally well in milk. An augmented system of culturing milk samples, which has given superior results in terms of the number of infected quarters detected, includes preculture incubation, then freezing of the milk sample, then inoculation of the medium with a larger than normal inoculum of milk.⁵⁰ The concern with augmented culture systems is that they may amplify contaminants obtained during sampling and therefore decrease the specificity of milk culture. Laboratory culturing techniques can be very time-consuming and expensive unless modern, prepackaged identification systems are used. They also provide the speed needed to make the examination a worthwhile one.

A milk sample is considered contaminated when more than three species of bacteria are isolated. A quarter is considered to be infected when the same bacteria is isolated in at least two out of three milk samples. A quarter is considered to be cured when bacteria, isolated at drying off, are not present in any samples 28 days after calving. An uninfected quarter at drying off that is infected at calving is considered to indicate a new intramammary infection. A quarter that is infected at drying off but infected with another bacteria at calving also indicates a new intramammary infection.

Selective culture plates, such as biplates (MacConkey agar and blood agar with 1% esculin), triplates (MacConkey agar, blood agar and TKT agar (thallium, crystal violet and staphylococcal toxin in 5% blood agar with 1% esculin)), Petrifilm® plates, which are selective culture media products,⁵¹ the MASTiK® diagnostic kit or the ColiMast® test can be used to differentiate between Gram-positive and Gram-negative pathogens and no growth,⁵² and may aid in the rational and targeted use of antimicrobial agents for clinical cases of mastitis. A commercially available cowside test for endotoxin (Limast-test®) is available in Scandinavia. The test takes 15 minutes to run on milk samples and is able to detect the presence of endotoxin using the Limulus amebocyte lysate assay. The test therefore can detect the presence or absence of Gram-negative bacteria but does not differentiate between E. coli and K. pneumoniae.⁵³ At least 10⁴–10⁵ cfu of Gram-negative bacteria are necessary for a positive test result; this does not decrease the clinical utility of the test, because low bacterial counts in the milk from infected quarters usually indicates that antimicrobial agents are not required in order to clear the infection.

There is interest in developing other cowside tests to determine whether the causative pathogen is Gram-negative or Gram-positive. One such approach uses dilution of the milk sample, filtration through a membrane with a pore size that retains bacteria, and staining of the bacteria with specific stains.⁵⁴ The filtration procedure reportedly takes 5 minutes, but the need for microscopic examination decreases the utility of this as a cowside test.

A common diagnostic problem is a bacteriologically negative culture in cows with clinical mastitis. Even when milk samples are collected appropriately and bacteriological culture is done using routine laboratory methods, 15–40% of samples from clinical mastitis episodes are bacteriologically negative (yield no growth). Failure of these samples to yield a mastitis pathogen may be the result of spontaneous elimination of infection, a low concentration of pathogens in the milk, intermittent shedding of the pathogen, intracellular location of the pathogens or the presence of inhibitory substances in the milk. Augmented culture techniques may reduce, but do not eliminate negative culture results and may facilitate growth of contaminant organisms. Dairy producers and veterinarians therefore face a dilemma when no bacteria or bacteria commonly regarded to be of minor pathogenicity, such as C. bovis or coagulase-negative Staphylococcus spp., are cultured from the milk of cows with clinical mastitis, particularly if clinical signs persist.⁵⁵ Most bacteriologically negative cases of clinical mastitis appear to be caused by low-grade infections with Gram-negative bacteria.⁵⁵ When no bacterial pathogen can be isolated from cases of clinical mastitis using standard culture techniques, enzyme-linked immunosorbent assays (ELISAs) may be used to detect antigens against S. aureus, E. coli, S. dysgalactiae, and S. agalactiae.⁵⁶ Antigens to these pathogens may be detectable using an ELISA in up to 50% of quarter samples from cows with clinical mastitis in which no pathogens were isolated but in which the SCC was more than 500000 cells/mL. Despite these promising findings, ELISAs are not widely used in the identification of mastitis pathogens.

The somatic cell count of composite or quarter samples

There is a strong relationship between the SCC of quarter samples of milk and the milk yield, with SCC increasing slightly as milk production decreases but increasing markedly with intramammary infection of the quarter. The distribution of SCC in a herd therefore reflects the distribution of intramammary infections. The most important factor affecting SCC in an individual cow is the number of quarters infected with a major or minor pathogen. In most herds, the prevalence of infection will increase through a lactation and will also increase with the age of the cow. Cell counts in the first few days of the lactation are often exceptionally high and unreliable as indicators of intramammary infection, and in uninfected cows the counts will drop to a low level within 2 weeks of calving and remain low throughout the lactation unless an intramammary infection occurs. The SCC of a cow that remains free of infection throughout her life will remain very low. However, older cows may have higher counts because the prevalence of infection is higher with age and older cows are more likely to have had previous infections with residual lesions and leaking of somatic cells into the milk. There are also consistent and significant differences in actual SCC between cows, individual cows tending to maintain the same class of count throughout their lives. Cows that have consistently low SCC do not seem to be more susceptible to mastitis than others. Attempts to base a breeding program to reduce the prevalence of mastitis on the selection of cows with an innately low composite SCC have been discarded because of fluctuations in numbers within cows.

Healthy quarters have a SCC below 100000 cells/mL, and this cutpoint should be used to indicate the absence or presence of intramammary infection on a gland basis. This cutpoint looks very solid for a gland, because many milk components differ from normal values whenever the SCC exceeds 100000 cells/mL. Moreover, mean SCC counts for bacteriologically negative quarters, quarters infected with minor pathogens and quarters infected with major pathogens were 68 000, 130000, and more than 350000 cells/mL, respectively.⁵⁸

Because of the time and labor saved it is now customary to do automated electronic cell counts on composite milk samples that have already been collected for butterfat testing. Regular reports of individual cow SCCs are therefore widely available in herds that routinely test production parameters of their cattle. An exciting new development in mastitis control is the portable somatic cell counter, which was designed for on farm use, thereby providing targeted and immediate SCC information for quarter or composite milk samples. Using the composite sample technique does distort the SCC; for example, the dilution of high-SCC milk from a bad quarter by low-SCC milk from three normal quarters could mean that a cow with one infected quarter might not be detected. Composite SCCs of less than 200000 cells/mL are considered to be below the limit indicative of inflammation, even though uninfected quarters have a SCC of less than 100000 cells/mL. Factors that affect the composite milk SCC include the number of infected quarters infected, the kind of infection (S. agalactiae is a more potent stimulator of cellular reaction than S. aureus), the strictness with which milk from cows with clinical mastitis is kept out of the bulk tank, the age of the cows (older cows have higher counts), the stage of lactation (counts are highest in the first days after calving and toward the end of lactation) and the herd’s average production, the cell count reducing as milk yield increases.⁵⁹

A SCC scoring system that divides the SCC of composite milk into 10 categories from 0–9, known as linear score, is becoming more widely used. The linear score is a base 2 logarithm of the SCC (in cells/mL), whereby linear score = log₂(SCC/100000) + 3. Likewise, to calculate SCC (in cells/mL) from the linear score (LS), the following formula is used: SCC = 100 000 × 2^{(LS – 3)}. A SCC of 100000 cells/mL therefore converts to a score of 3. Each 1-unit increase (or decrease) in linear score is associated with a doubling (or halving) of the SCC. For example, score 2 is equivalent to a SCC of 50000 cells/mL, and scores of 4 and 5 correspond to 200000 and 400000 cells/mL. Conversion of SCC to linear score values is performed as shown in Tables 15.3 and 15.4.. The principal reason for using the linear score is to achieve properties that are required in order to use conventional statistical methods: mean equal to median, normal distribution and uniform variance amongst samples within lactation, amongst cows within herd or among daughters within sire.

Table 15.3 Linear score calculation from the somatic cell count

Table 15.4 Conversion of linear scores to somatic cells counts (cells/mL) and predicted loss of milk⁹⁸

The proportion of neutrophils in the SCC is very low (<11%) in healthy quarters but is markedly increased in quarters with intramammary infection (to >90%). Accordingly, the percentage of neutrophils in the SCC may provide a useful indication of intramammary infection, but is not currently performed.⁵⁸

Somatic cell counts can also be determined for colostrum, where they are useful in indicating the presence of intramammary infection (Table 15.2).⁶⁰

Table 15.2 California Mastitis Test reactions and equivalent somatic cell counts (SCCs) and linear scores for bovine milk and SCCs for bovine colostrum

The California Mastitis Test of quarter samples

The CMT is the most reliable and inexpensive cowside test for detecting subclinical mastitis. The CMT is also known as the Rapid Mastitis test, Schalm test or Mastitis-N-K test, was developed in 1957 and constituted a modification of the Whiteside test. The CMT reagent contains a detergent that reacts with DNA of cell nuclei, and a pH indicator (bromcresol purple) that changes color when the milk pH is increased above its normal value of approximately 6.6 (mastitis increases pH to 6.8 or above). The CMT is mixed with quarter milk samples that have been previously collected into a white container and the sample is gently swirled; the result is read within 15 seconds as a negative, trace, 1, 2, or 3 reaction depending on the amount of gel formation in the sample. Maximum gel formation actually occurs from 1–2.5 minutes, depending on the quarter SCC and continued swirling of the mixture after the time of peak viscosity produces an irreversible decrease in viscosity.⁶¹ Cows in the first week after calving or in the last stages of lactation may give a strong positive reaction.

The close relationship between the CMT reaction and the SCC of milk and the reduced productivity of affected cows is shown in Table 15.4. If the CMT is used to minimize the false negative rate (produce the highest sensitivity), then the test should be read as negative (CMT = negative) or positive (CMT = trace, 1, 2 or 3). If the CMT is used to minimize the false-positive rate (produce the highest specificity) for culling decisions, then the test should be read as negative (CMT = negative or trace) or positive (CMT = 1, 2 or 3).

CMT scores can also be determined for colostrum, where the score is useful in indicating the presence of intramammary infection (Table 15.2). The equivalent SCC for CMT scores of negative or trace are different for colostrum and milk, but the SCC for CMT scores of 1, 2 and 3 are similar for colostrum and milk.⁶²

The NAGase test of composite or quarter samples

The NAGase test is based on the measurement of a cell-associated enzyme (N-acetyl-β-d-glucosaminidase) in the milk, a high enzyme activity indicating a high cell count. NAGase is an intracellular lysosomal enzyme derived primarily from neutrophils but also from damaged epithelial cells. The test is suited to the rapid handling of large numbers of samples because of the ease of its automation, and the test can be done on fresh milk and read on the same day.⁶³ However, because most of the NAGase activity is intracellular, samples should be frozen and thawed before analysis to induce maximal NAGase activity.⁵⁸ The NAGase test is reputed to be the most accurate of the indirect tests and as good as SCC in predicting the infected status of a quarter. The NAGase test uses a less sophisticated reading instrument than the average automatic cell counter. If all tests are available it is best to consider the NAGase test and SCC as complementary tests and carry out both of them. Milk NAGase levels are high at the beginning and the end of lactation, as with cell counts. The test has also been validated for use with goat milk.

Electrical conductivity tests of quarter samples

A test that has received a lot of attention because it can be used in robotic milking systems is based on the increase in concentration of sodium and chloride ions, and the consequent increase in electrical conductivity, in mastitic milk.⁶⁴ The electrolyte changes in milk are the first to occur in mastitis and the test has attractions for this reason. A number of factors affect these characteristics, however, and to derive much benefit from the test it is necessary to examine all quarters and use differences between the quarters to indicate affected quarters. For greater accuracy all quarters need to be monitored each day. An experimental unit that takes all these factors into consideration has been fitted to a milking machine and, by a computer-prepared analysis, monitors variations in electrical conductivity in each quarter every day.⁶⁵ Electrical conductivity is attractive as a test because it measures actual injury to the udder rather than the cow’s response to the damage, as SCC and NAGase activity do.⁶⁶ However, a meta-analysis indicated that using an absolute threshold for conductance did not provide a suitable screening test, as both sensitivity and specificity were unacceptably low.⁶⁷ The use of differential conductivity (within cow quarter comparison) results in improvement in test sensitivity and specificity, and is currently the only recommended application of this indirect test.^58,68

The most commonly promoted method for measuring electrical conductivity is a hand-held device with a built-in cup into which milk is squirted (foremilk is preferred). Experimentally induced clinical mastitis due to S. aureus and S. uberis was detectable by changes in electrical conductivity of foremilk: 90% of cases were detectable when clots first appeared and 55% of cases were detectable up to two milkings prior to the appearance of clots.⁶⁴ This suggests that clinical mastitis associated with these two major pathogens may be able to be detected earlier by electrical conductivity than by waiting for milkers to detect visible changes in the milk.

Comparison of indirect methods

The effects of subclinical intramammary infection on several parameters in foremilk from individual quarter milk samples have been compared.⁶² Somatic cell count, electrical conductivity, pH, NAGase activity and the concentrations of sodium, potassium, lactose and alpha-1-antitrypsin were measured from individual quarters. Somatic cell count, NAGase activity, electrical conductivity and concentrations of sodium, alpha-1-antitrypsin and lactose were all useful indirect indicators of infection. The SCC was better able to discriminate between infected and uninfected quarters and cows than were the electrical conductivity, pH and NAGase activity.

Is antimicrobial therapy indicated?

The first decision is whether to treat a particular case with antimicrobial agents and whether supportive therapy is required. Therapy decisions should be made in context with the overall objectives of the lactating cow treatment protocol. The availability of approved, effective treatment products is an essential component of the program. A number of factors are important in determining which cases of mastitis should be treated during lactation. These factors include the type of pathogen involved, the type and severity of the inflammatory response, the duration of infection, the stage of lactation, and the age and pregnancy status of the cow.

Which route of administration (intramammary, parenteral or both?)

The second decision is the route of administration. The goal of antimicrobial treatment is to attain and maintain an effective concentration at the site of infection. Three pharmacological compartments are recognized for infection by mastitis pathogens:

Table 15.5 Summary of three-compartment model for anatomical location of infection due to mastitis pathogens in cattle

In general, infections confined primarily to the milk and ducts (such as C. bovis, coagulase-negative staphylococci) are easily treated with intramammary antibiotics. In contrast, infections due to mastitis pathogens with potential for systemic infection (such as E. coli, K. pneumoniae, M. bovis) are best treated with parenteral antibiotics. Mastitis pathogens that are the most difficult to treat are those that are principally infections of parenchymal tissue (such as S. aureus, A. pyogenes); this is because it is more difficult to attain and maintain an effective antibiotic concentration at this anatomical site when administering antibiotics by the intramammary or parenteral routes.

Which antimicrobial agent should be administered?

The third decision is the antimicrobial agent. The selection of the antimicrobial class for the particular mastitis pathogen has traditionally been based on culture and susceptibility testing and, although some in vivo data are now available, the choice is still largely dependent on case studies rather than on controlled experiments. Culture and antimicrobial susceptibility testing of the pathogen is not necessarily a justifiable basis for selecting the antimicrobial agent to be used in individual cows, and the response to treatment of clinical mastitis in two recent studies^84,85 was unrelated to the results of in vitro susceptibility tests.

Antimicrobial agents are usually selected based on availability of labeled drugs, clinical signs in the cow, milk culture results for previous mastitis episodes in the herd, experience of treatment outcome in the herd, treatment cost and withdrawal times for milk and slaughter. Many veterinarians and researchers have also recommended the use of susceptibility testing to guide treatment decisions. The validity of agar diffusion susceptibility breakpoints derived from humans in the treatment of bovine mastitis has not been established and is extremely questionable because bovine mastitic milk pH, electrolyte, fat, protein and neutrophil concentrations, growth factor composition and pharmacokinetic profiles differ markedly from those of human plasma.⁸⁶ Moreover, antibiotics are distributed unevenly in an inflamed gland, and high antibiotic concentrations can alter neutrophil function in vitro and therefore have the potential to inhibit bacterial clearance in vivo.

Adequate databases of in vitro minimum inhibitory concentration (MIC) values for clinical mastitis pathogens are currently unavailable, although adequate databases are available for subclinical mastitis isolates. Although we have a good knowledge of the pharmacokinetics of many parenteral antibiotics used to treat clinical mastitis, most pharmacokinetic data have been obtained in healthy cattle and it has not been determined whether pharmacokinetic values in healthy cows are the same as those in cows with clinical mastitis. In addition, pharmacokinetic values for many of the intramammary antibiotics used to treat clinical mastitis are unknown, and we have a limited understanding of the pharmacodynamics of antibiotics in treating mastitis. More importantly, the breakpoints currently recommended for all parenterally and almost all intramammarily administered antibiotics are based on achievable serum and interstitial fluid concentrations in humans after oral or intravenous antibiotic administration. The relevance of these breakpoints to achievable milk concentrations in lactating dairy cows after intramammary, subcutaneous, intramuscular or intravenous administration is dubious at best.⁸⁶

Results from field studies are available to evaluate the validity of susceptibility breakpoints in guiding treatment of cows with clinical or subclinical mastitis. The results from these field studies suggest that the following antibiotics may have valid (but not necessarily optimal) breakpoints for treating clinical or subclinical mastitis associated with specific bacteria: parenteral penicillin G for subclinical S. aureus infections, intramammary cephapirin for clinical Streptococcus spp. infections, and parenteral trimethoprim– sulfadiazine for clinical E. coli infections. Of these three antibiotics, the breakpoints for penicillin G and cephapirin have only been validated for bacteriological cure, whereas the breakpoint for trimethoprim– sulfadiazine is validated for clinical cure.⁸⁴ Because duration of infection before treatment, antibiotic dosage, dosage interval and duration of treatment influence treatment outcome, many more field studies must be completed to validate the currently assigned antibiotic breakpoints for pathogens causing clinical mastitis.

To properly utilize the known pharmacokinetics of parenterally and intramammarily administered drugs, it is necessary to know something about their diffusion into mammary tissue, the degree of binding of a drug to mammary tissues and secretions, the ability to pass through the lipid phase of milk and the degree of ionization. All of these factors influence the level of the antibiotic in the mammary gland. For lactating cows the preferred treatment is one that maintains a MIC for 72 hours without the need for multiple infusions and without prolongation of the withdrawal time. The most successful antimicrobial agents for dry period treatment are those that persist longest in the udder, preferably as long as 8 weeks. These characteristics depend on the release time from the transport agent in the formulation, and the particle size and diffusion capabilities of the antibiotic.

The formulation of the preparation will affect the duration of the maintenance of the MIC. The third-generation cephalosporins (such as ceftiofur) and fluoroquinolones are the drugs of choice for use in cases in which the infection may be associated with either a Gram-positive or Gram-negative organism; however, these antimicrobial agents may not be able to be used to treat mastitis in some countries. Mixtures of penicillin and an aminoglycoside are also in common use for this purpose. Penicillin G and penethamate are favored for Gram-positive infections.

Of special importance are the beta-lactamase-producing strains of S. aureus, against which beta-lactam penicillins are ineffective; cloxacillin is a commonly used and effective intramammary formulation for these strains of S. aureus. The drugs that have the best record of diffusion through the udder after intramammary infusion are penethamate, ampicillin, amoxicillin, erythromycin and tylosin. Those of medium performance are penicillin G, cloxacillin and tetracyclines. Poor diffusers, which have a longer half-life in the udder because they bind to protein, include streptomycin and neomycin. Streptomycin is not much used now because of the high level of resistance to it, especially by S. uberis and E. faecalis.

What should be the frequency and duration of treatment?

The fourth decision is the frequency and duration of treatment. The frequency of administration for parenterally administered antimicrobial agents is dependent primarily on their pharmacokinetics and pharmacodynamics. Fluoroquinolones and aminoglycosides are concentration-dependent antimicrobial agents where increasing concentrations at the site of infection increase the bacterial kill rate. Macrolides, beta-lactams, and lincosamides are time-dependent antimicrobial agents where exceeding the minimum inhibitory concentration at the site of infection for a prolonged percentage of the interdosing interval correlates with improved efficacy.⁸⁷ In contrast, the frequency of administration for intramammary formulations is dependent primarily on the milking schedule, in that these agents are primarily cleared by milk removal. For example, the clearance of pirlimycin is strongly and positively correlated (r = 0.97) to 24-hour milk production at the time of dosing.⁸⁸ With all intramammary formulations being licensed based on the results of studies of twice-daily milking, the recent industry trend in some parts of the world towards thrice-daily milking has created uncertainty as to whether intramammary treatment should be repeated after every milking, or even whether once-a-day intramammary administration is as efficacious as twice- or thrice-daily administration.

Recent studies have confirmed long held beliefs that appropriate antimicrobial therapy (commonly called extended or aggressive antimicrobial therapy) for 5–8 days is much more effective in treating intramammary infections than label intramammary therapy (2–3 d). In other words, increasing the duration of antimicrobial administration increases treatment efficacy.^89-92 Extended antimicrobial therapy is opposed by producers because such treatment may be off-label and results in a longer milk withhold time, and consequently the amount of milk that has to be discarded. Extended therapy is opposed by dairying administrators because of the inevitable increase in the number of infringements of health regulations relating to antibiotic residues in milk. The inappropriately short treatment duration for most intramammary products has been a major hindrance to developing effective antimicrobial treatment protocols.

Infusion procedure

The teats must be cleaned and sanitized before infusing the quarter in order to avoid introduction of infection. The following steps are recommended:

Diffusion of infused intramammary drugs is often impeded by the blockage of lactiferous ducts and alveoli with inflammatory debris. Complete emptying of the quarter by the parenteral injection of oxytocin (10–20IU intramuscularly) followed by hand-stripping of affected quarters before infusion has been recommended in cases of clinical mastitis, but efficacy studies are lacking, the volume stripped is usually small and the procedure is painful to the cow. If stripping is performed, the intramammary infusion is given after the last stripping of the day has been done, avoiding any further milking of the gland until the next milking. Our current knowledge can be summarized by the following: ‘To strip or not to strip, that is the question.’

Emergency treatment when the type of infection is unknown

Cases of acute and peracute mastitis (abnormal cow) in lactating cows, and in dry cows close to calving, are serious problems for the field veterinarian. The need for treatment is urgent; it is not possible to wait for the results of laboratory tests to guide the selection of the most appropriate antibiotic. Clinical findings, season of the year and management practices may give a broad hint as to the specific bacterial cause, but in most such circumstances it is necessary to use a broad-spectrum approach to treatment.⁹⁵ Parenteral therapy with oxytetracycline (administered intravenously to increase bioavailability and therefore plasma and milk concentrations), a potentiated sulfonamide or similar broad-spectrum antimicrobial agent should be supplemented with intramammary infusion with a beta-lactamase-resistant antimicrobial such as a first-generation cephalosporin (cephapirin), a third-generation cephalosporin (e.g. ceftiofur), penicillin G–neomycin combination or other approved broad-spectrum intramammary infusion.⁹⁶ Parenteral ceftiofur is not effective in clinical mastitis episodes that have abnormal secretion or abnormal gland and secretion.⁹⁷

Field studies show that, in herds in which clinical mastitis is often caused by environmental pathogens, intravenous administration of oxytetracycline, intramammary infusion of cephapirin and supportive therapy (including intravenous administration of flunixin meglumine or fluids) produces a higher rate of clinical and bacteriologic cure than supportive treatment alone.⁹⁶ In addition, antimicrobial treatment is more effective than supportive treatment alone.⁸⁶ In cows with clinical mastitis caused by E. coli the use of procaine penicillin G IM was no more effective than not using antimicrobial agents;⁹⁸ this result is expected based on penicillin’s Gram-positive spectrum of activity. Knowledge of the likely causative agent is therefore helpful when making decisions about therapy of clinical mastitis episodes during lactation.

Provision of other supportive therapy such as fluids and electrolytes is also crucial to the survival of the cow and minimization of the severity of the mastitis and extent of permanent injury to the udder. The efficacy of frequent stripping, with or without intramammary infusion, is uncertain. NSAIDs decrease pain associated with an abnormal gland; in addition, they enhance recovery and reduce fever in severe cases.

Treatment when the infecting organism is known

A common situation encountered by a bovine practitioner is the dairy herd that has had an outbreak of clinical mastitis or has received a warning notice from the milk processor that the bulk milk SCC or bacterial count is above acceptable limits. The situation calls for a complete mastitis control program, including conducting an investigation to determine the causative bacteria present, the source of the infection, hygiene in the milking parlor and the importance of risk factors such as milking machine management, plus recommended antimicrobial preparations selected on the basis of the causative agent. Treatment of a number of identified subclinical cases at the commencement of the program, and of individual cases subsequently, can be based on the known common infection in the herd. Among Gram-positive cocci, the response to antimicrobial agents is excellent for streptococci. For staphylococci a cure rate of 65% is about the best that can be expected, and unless there are good reasons for doing otherwise it is recommended that treatment be postponed until the cow is dry. Standard treatments for lactating cows include penicillin alone (100000 units) or in combination with streptomycin (1 g) or neomycin (500 mg), and a combination of ampicillin (75 mg) and sodium cloxacillin (200 mg). Acid-resistant penicillins, e.g. phenoxymethylpenicillin, are probably best not used as mammary infusions because of their ability to pass through the human stomach, thus presenting a more serious potential threat to humans drinking contaminated milk. Because of the widespread and often indiscriminate use of penicillin, a large part of the mastitis that occurs is associated with penicillin-resistant bacteria, especially S. aureus. Treatment programs need to take this into account when recommendations are made about the antibiotic to be used.

Intramammary infections associated with environmental streptococci that manifest signs of clinical mastitis are usually acute but only moderately severe. In most of these cases the streptococci are sensitive to antimicrobial agents, and they often recover spontaneously with good management and nursing care. If not, they usually respond well to therapy. Bacteriological cure rates of 60–65% can be expected following a single intramammary infusion of a cephalosporin product. In one randomized controlled field trial of clinical mastitis associated with Streptococcus spp. or coliform bacteria, the clinical cure rate by the tenth milking was significantly higher when intramammary cephapirin, intravenous oxytetracycline, or both, were used along with supportive therapy (oxytocin and stripping of affected glands and, in severely affected cows, the use of flunixin meglumine and fluids) compared to supportive treatment alone.⁹⁹ These results indicate that, in herds in which clinical mastitis is often associated with environmental pathogens, antimicrobial therapy and supportive therapy may result in a better outcome than supportive therapy alone.

Treatment of subclinical mastitis

It is generally considered not advisable to treat subclinical mastitis during lactation. However, it is important to consider the causative organism and the udder health status of the herd. There are several situations in which lactational therapy of subclinical mastitis is indicated; for example, herds with S. agalactiae infections should consider several approaches to therapy during lactation. S. agalactiae infections respond well to therapy during lactation, with cure rates of 80–100% expected.⁵⁹ All approved intramammary therapy preparations are efficacious, including penicillin, cephalosporins, cloxacillin and erythromycin. In herds with a high prevalence of S. agalactiae mastitis, blitz therapy can be used for eradication of the pathogen, increased milk production and reduced penalties for high SCCs. There is, however, a risk of residue violation, problems with disposal of milk from treated cows and considerable costs involved. It is also important to insure that standard mastitis control procedures, such as postmilking teat disinfection and blanket dry cow therapy, have been implemented. The benefit:cost ratios for various approaches to blitz therapy of S. agalactiae infected herds have been studied.⁵⁹ The prevalence of infected cows, and their stage of lactation, are important determinants of the type of program selected.

Therapy of cows with subclinical mastitis due to S. aureus during lactation is much less rewarding. Under field conditions, cases of S. aureus are difficult to cure during lactation. Reported cure rates following intramammary therapy are between 15% and 60%. Lactational therapy of subclinical S. aureus mastitis using intramuscular penicillin along with intramammary amoxicillin infusion, compared with the intramammary infusion alone, increased the cure rate to 40%, which represented a doubling of the cure rate with intramammary therapy alone.⁴⁸ If treatment by this method is used in combination with data on the age of cow, stage of lactation, duration of infection and level of SCC, the economic benefit of treating some cases of S. aureus mastitis during lactation may be attractive.

Subclinical infections associated with environmental streptococci, and occasionally by coliform organisms, can be found in moderate numbers in some herds.⁵⁶ Although spontaneous cure rates are higher with these environmental infections, individual cows may merit treatment during lactation. In these cases, the previously listed factors should be used, and are important in the selection of cases to be treated.

Prepartum antibiotic treatment of heifers is of benefit in herds experiencing a high incidence of clinical mastitis in recently calved heifers. Coagulase-negative staphylococci are frequently isolated from late-gestation heifers, and intramammary treatment with sodium cloxacillin (200 mg) or cephapirin sodium (200 mg) 7 days before expected parturition is highly effective and economically beneficial.¹⁰⁰

Antimicrobial residue tests

Several cowside tests are available to detect antimicrobial residues in the milk of cows that have been treated for mastitis.¹¹³ The goal of cowside testing is to assist in the production of high-quality, antimicrobial-residue-free milk from dairy herds. To be consistent with the intent of a quality assurance program, cowside testing would be used only on cows recently treated with antimicrobial agents and only after appropriate milk withholding times had been followed. The ideal test would have a high sensitivity and high specificity.

Most of the cowside screening tests for antimicrobial residues are imperfect because of a high rate of false-positive results when used on field samples. The direct costs to producers can be high because of the unnecessary disposal of milk and imposition of fines and penalties. False-positive results also cause the unnecessary culling of some cows, and concern about the interpretation of positive assay results, the appropriateness of withholding periods and the safety of milk creates mistrust among consumers, producers, veterinarians and regulatory personnel. The specificity of four commercially available tests ranged from 0.78–0.95.¹¹⁴ None of the test kits has been validated to meet performance standards for sensitivity and specificity. This applies to individual cow samples, bulk tank milk samples and tanker truck samples.

The presence of naturally occurring bactericidal products in the milk of cows with acute and convalescent mastitis is the most likely cause of the false-positive results of the tests (such as Delvo-test®) that are based on bacterial growth inhibition of beta-lactam antimicrobial agents.¹¹³ Immunoglobulins, complement, lysozyme, lactoferrin and phagocytic cells are products of inflammation in the milk of cows with mastitis that can inhibit bacterial growth. The milk from cows with experimental endotoxin-induced mastitis is at increased risk for false-positive assay results using commercial residue tests.¹¹⁴ The incidence of false-positive results is very low in milk from cows that have not had a history of mastitis or antimicrobial therapy.¹¹⁵ Naturally occurring bactericidal products in mastitic milk can be removed by heating at 82°C for 5 minutes; this temperature does not denature antimicrobial agents present in milk.¹¹⁶ Heat treatment therefore appears to provide a very practical way to reduce false-positive results on milk from individual cows.

A sample of milk can be submitted for antimicrobial residue testing up to three times. First, a producer may test a sample from a specific cow at the end of her withdrawal period. Second, milk is sampled at the tanker truck level. Third, should the tanker truck sample have positive results, bulk tank milk samples from each dairy herd that contributed to that tanker truck are tested.

There is a need for validation of the diagnostic assays used to detect antimicrobial residues in milk.¹¹⁷ Acceptance of assays for regulatory purposes must be based on protocols that include field estimates of assay performance before the assays are used by the public. Three strategies have been suggested to balance public health concerns with economic concerns of dairy producers caused by false-positive results:

It is suggested that regulatory monitoring of residues at a national level will be best served by use of a combination of at least two assays: initial screening with a highly sensitive and inexpensive assay followed by confirmation testing with an assay of high specificity (> 99%) that can quantify the concentration of the antimicrobial residue. All tanker samples that yield positive results with a screening assay should be rechecked with a quantitative assay. If the quantitative assay detects a concentration greater than the safe level, safe concentration or tolerance level, only then would the milk be deemed to have violative residue and fines and penalties be imposed.¹¹⁷ The complex dynamics of current milk residue tests discourage practitioners from recommending testing procedures to dairy producers.¹¹⁸

As an approximate guide, the recommended periods for which milk should be withheld from sale after different methods of antimicrobial administration are (in times after last treatment):