Identification of Etiologic Agents

Effective treatment and control of mastitis is based on knowl­edge of the etiology of the infection. As dairy farms increase in size, an increasingly diverse group of pathogens have been associated with the occurrence of mastitis.^6,142-144 Clinical signs may be suggestive of some pathogens, but it is impossible to diagnose the etiology based on the appearance of the milk, gland, or animal.

MILK SAMPLES COLLECTED FROM INDIVIDUAL ANIMALS. Milk samples may be individually collected from affected quarters (quarter milk samples) or combined from all four glands into a single vial (composite milk samples). Quarter milk samples collected from affected glands are more sensitive in detection of bacteria from subclinical infections as compared to composite milk samples, although the sensitivity of composite samples increases with the number of infected glands per cow.¹⁴⁸ Repeated sampling increases the likelihood of detecting glands that are intermittently shedding some pathogens.⁵ The con­centration of bacteria per milliliter of milk varies among pathogens. Some pathogens (such as most Streptococcus spp.) consistently shed large numbers of bacteria in milk. For these pathogens, the use of composite milk samples may be acceptable because concentrations of bacteria will likely consistently exceed the microbiological detection limit.¹⁴⁹ For many other pathogens (such as S.

Microbiological culture or polymerase chain reaction (PCR) testing of composite milk samples is often used to reduce the cost of routine screening of high-risk animals for the presence of existing subclinical infections with potentially contagious mastitis pathogens. Pooling 5 to 10 aseptically collected composite or quarter milk samples is another strategy that is used in some herds to further reduce costs. When either of these strategies is used, it is important to recognize that the potential for false negative outcomes is increased. The sensitivity of composite milk samples for detection of S. aureus and Streptococcus dys- galactiae was estimated to be about 73% to 77% in contrast to about 60% to 62% for Streptococcus uberis and non-aureus staphylococci (NAS).¹⁵¹ In all instances, sensitivities increase as the number of infected mammary gland quarters increases.¹⁵¹ When using pooled or composite samples, veterinarians should routinely review cow histories to identify cows with increased SCCs that are typical of subclinical infections.

Mastitis is usually caused by a single type of bacteria, and the National Mastitis Council has developed standards that specify that recovery of more than two colony types of bacteria cultured from an aseptically collected milk sample is indicative of contamination.⁵ Before collection of milk samples, technicians should be trained in techniques that ensure aseptic collection of the sample; adherence to aseptic sampling procedures is even more critical when DNA-based diagnostic tests are used. To maximize the possibility of recovering bacteria from the sample, milk should be collected after the teats have been prepared for milking (application of premilking disinfectant and drying) but before the units are attached.¹⁵² Collection of samples before milking is usually easier because cows are more likely to stand still and there is less pressure from farm workers to release the cows.

The cap should be removed from the sample vial without touching the inside and held so that the inner surface faces down. Milk from the teat to be sampled can be directed at an angle into the sampling vial. A sample size of 3 to 5 mL is usually adequate. The cap should be replaced immediately after the sample is obtained. When sampling multiple teats, the far teats should be scrubbed before the near teats and the near teats sampled first, to avoid contamination by the sampler's arm or hand. Samples must be cooled immediately, refrigerated at 4° C, held on ice, or frozen until cultured. Failure to use aseptic technique or mishandling after collection can produce

TABLE 36.2

Summary of Characteristics of Common Mastitis Pathogens

IMM, Intramammaty; SCC, somatic cell count.

ιυ Ul

erroneous results. If samples are to be submitted to a diagnostic laboratory, they should be submitted within 24 hours of col­lection. If samples cannot be processed within 24 hours, they should be frozen until transported to the lab. Freezing for periods of more than 2 weeks has minimal effects on recovery of most mastitis-causing bacteria but can reduce recovery of Mycoplasma spp.¹⁵3

When proper sampling and laboratory techniques are used, the recovery of bacteria from bovine milk samples is highly specific for mastitis. However, microbiological examination of milk obtained from glands affected with clinical or subclinical mastitis is not very sensitive.¹¹⁴ When a single milk sample is obtained from dairy cattle exhibiting clinical or subclinical mastitis, approximately 35% to 50% of milk samples will be culture negative (see Table 36.1).^5,6 Repeated sampling of affected quarters can increase the probability of reaching a diagnosis.⁵,¹⁵⁰

Failure to recover bacteria from a milk sample can be a result of either a successful or a failed inflammatory response. For example, the inability to culture viable bacteria from milk samples collected from quarters with chronically high SCCs may be a result of suppression of bacterial growth caused by the chronic inflammatory response. In this instance, ongoing inflammation is likely caused by chronic bacterial infection and is indicative of failed inflammatory response. In contrast, failure to recover viable bacteria from milk samples obtained from cows with clinical mastitis is often due to spontaneous clearance of bacteria prior to onset of visible clinical signs. Many of these culture-negative clinical cases are the result of successful inflammation, and if the clinical signs are not severe, most of these cows have excellent prognoses without use of antimicrobial therapy.¹⁴⁰

Laboratory Methods. Standardized laboratory methods for identification of mastitis pathogens have been well defined by the National Mastitis Council.⁵ In general, laboratory technicians inoculate media that contain nutrients required for bacterial growth, and the inoculated media are placed in an incubator that contains the appropriate atmosphere and temperature to encourage growth of the target organisms. After 24 to 48 hours, the plates are examined for growth, and the bacteria are identified using a variety of specific test methods that depend on observable characteristics of the bacteria. In most mastitis laboratories, 10 to 100 μL of each milk sample are inoculated onto a portion of a blood agar plate (5%). The inoculum volume determines the lower limit of detection. For example, one colony observed on a plate inoculated with 0.01 mL (10 μL) is equivalent to approximately 100 CFU/mL of milk, whereas one colony observed using a 0.1-mL (100-μL) inoculum is equivalent to approximately 10 CFU/mL. The use of larger (0.05- or 0.10-mL) inocula increases recovery rates for pathogens shed in low concentrations, such as E. coli or S. aureus. Other ways to increase recovery are pre-culture centrifugation,¹⁵⁴ incubation,¹55 or freezing¹⁵' of the milk, or a combination of these methods. The optimal method is pathogen dependent. Augmented methods are most useful for detecting subclinical S. aureus infections and reducing false­negative results for clinical mastitis samples.

After inoculation, agar plates are incubated at 37° C and observed for growth at 24 and 48 hours.⁵ Although there is no absolute definition of IMI,¹⁵⁸ the presence of at least 100 to 300 CFU/mL is usually required to define an infection.^5,114 Identification of bacteria is made based on phenotypic char­acteristics of the colonies and the result of additional laboratory tests. S. aureus is usually differentiated from other staphylococci based on a positive coagulase reaction and other typical phenotypic characteristics, such as hemolysis.⁵ Streptococci are usually identified using the Christie-Atkins-Munch-Petersen (CAMP) test and esculin reactions. When milk samples originate from cases of clinical mastitis, MacConkey agar is usually also inoculated to facilitate the rapid identification of gram-negative, lactose-fermenting organisms (coliforms). Classic microbiologi­cal techniques use additional biochemical tests for final identification at the species level, but other techniques for identification of mastitis pathogens are increasingly used. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to increase accuracy of diagnosis of NAS and streptococci.^159,160 Identifica­tion of Mycoplasma spp. requires the use of media containing specific nutrients not found in general media and incubation in a carbon dioxide-enhanced environment.

On-Farm Culture. As farm size has increased and special­ized workers have become more common, on-farm culture (OFC) programs have been developed.¹⁶¹ Methods used for OFC are not usually the same as those used in professional diagnostic laboratories and are often performed by farm personnel who may have some technical expertise but lack formal training in microbiological methods. On most farms, OFC methods are based on the use of laboratory shortcuts and have a goal of rapidly reaching a presumed diagnosis to guide treatment.^162-164 The most basic method of OFC is the use of biplates, triplates, or quadplates that contain selective¹⁶⁵ agars or chromogenic media.¹⁵⁴ Growth on a selective agar is used to differentiate cases caused by gram-positive or gram­negative bacteria, culture-negative cases, or in some instances specific pathogens. After 24 hours of incubation, culture plates are observed, and the treatment protocol is specified based on the culture outcome.^140,163,166 Typical agars that are used include MacConkey agar (selective for growth of gram-negative bacteria); TKT agar (selective for growth of streptococci), and Factor, Baird-Parker, or KLMB media (selective for growth and differentiation of some staphylococci). Studies have indicated that using selective media in OFC systems is about 80% accurate in differentiating gram-positive and gram-negative pathogens as compared to diagnostic labora- tories.¹⁶⁵ The use of OFC to make more specific pathogen diagnoses is not as accurate and requires additional training of personnel.

Most smaller herds do not have sufficient cases of mastitis to develop the expertise needed for OFC, and one alternative is to offer in-veterinary clinic culturing (IVCC). In these instances farmers usually collect a milk sample and immediately initiate treatment, but they may stop or modify treatment duration or the drug after receiving a preliminary microbiologi­cal diagnosis from their veterinarian within 24 hours. Develop­ment and oversight of a culture program (either OFC or IVCC) is an ideal way for veterinarians to increase involvement in mastitis control programs. Using veterinary technicians to supervise these programs may also increase veterinary involve­ment and oversight of mastitis treatments. When OFC is used, veterinary technicians can visit farms to restock supplies, train farm personnel, and provide oversight and quality control.

MICROBIOLOGICAL ANALYSIS OF BULK TANK MILK. Micro­biological examination of bulk tank milk samples is performed using a variety of laboratory techniques depending on the objective of the testing.^5,106 Enumeration of the total number of aerobic bacteria in bulk milk is routinely performed by milk processors as part of the regulatory requirements for milk production. Standardized methods for counting bacteria may be based on culture or performed using automated counting of stained bacterial cells.¹¹⁹ Processors also commonly perform additional laboratory tests to assess hygienic conditions of milk harvest and storage and may use results of these tests to determine premium payments. Comparison of the number of psychotrophic, thermoduric, or coliform bacteria from various milk samples can be used to identify problems with dirty udders, inadequate premilking preparation, cleanliness of milking equipment, or cooling stored milk.⁵ Methods used for these tests are simply quantitative and do not result in identification of specific microorganisms.

Qualitative bulk tank cultures (BTCs) are usually performed to identify specific pathogens shed by cows that are being milked while affected with subclinical mastitis.^5,106 Methods of sampling, laboratory techniques, and reporting formats have not been standardized, thus it is difficult to compare results of BTCs among laboratories. Because of the variation in laboratory methods, it is best to submit bulk tank milk samples to a specialized laboratory that tests several dilutions using selective media for isolating and counting colonies. The laboratory should be informed if identification of Mycoplasma spp. is of interest so that the proper media and incubator conditions are used.

The interpretation of results of BTCs can be confusing, as bacteria can originate from teat skin, subclinical mastitis infec­tions, failure of milking technicians to exclude milk from cows that have clinical mastitis, poor hygienic conditions, failure to adequately cool milk, and use of inadequately cleaned milking equipment. Because there are numerous opportunities for bacteria to contaminate bulk milk, interpretation of BTCs is based on knowledge of reservoirs of the organisms found in the milk.^5,167 Although few scientific studies have been performed to validate criteria for interpretation of BTCs, suggested interpretive criteria have been published.⁵ A few organisms (S. agalactiae, S. aureus, and Mycoplasma bovis) are known to originate primarily from infected cows, but many organisms have multiple potential sources. Organisms such as NAS may originate from IMI or from poorly disinfected teat skin and udders that have been contaminated by bedding sources. Non-agalactiae streptococci are commonly found in the dairy farm environment and are associated with dirty udders, but these organisms can also cause subclinical mastitis. The natural duration of subclinical IMI caused by E. coli is short, and there are multiple environmental sources of this organism.¹¹⁶ Thus when many E. coli are found in bulk milk, the most likely source is contamination during the milking process.¹⁶⁸ When organisms that have environmental niches are found in bulk tank milk, sources other than IMI should be investigated before assuming that infected cows are the source.

The sensitivity of a single BTC for detection of herds with infected cows is generally considered to be low, and results should not be used to estimate prevalence of infected cows.⁵ Positive predictive values of BTCs (detection of at least one positive cow when the organism was found in a BTC) are estimated to be more than 97% for S. agalactiae and S. aureus and more than 80% for Mycoplasma spp.¹⁶⁹ Due to low sensitivity, the inability to isolate selected mastitis pathogens from bulk milk does not indicate the absence of subclinically infected cows, and negative BTCs routinely occur in large herds that contain infected cows. The relatively low sensitivity of a single BTC for screening herds for contagious pathogens can be improved by using enhanced laboratory techniques or by increasing the frequency of sampling. The use of commingled samples that have been independently collected for 3 to 5 consecutive days is recommended to reduce false-negative results that occur as a result of variation in bacterial shedding.¹⁶⁷

MOLECULAR TESTING. The continued development of molecular techniques has the potential to improve diagnosis of mastitis; however, a full review of these technologies is beyond the scope of this chapter. A variety of molecular methods are used to diagnose mastitis pathogens, identify potential zoonotic agents in milk, identify genetic diversity of resistance genes, and investigate mastitis outbreaks and milk quality problems.^170-173 Some molecular methods are used to identify specific strains of bacteria to determine the likely source or transmission of mastitis pathogens.^174,175 Other methods are used to improve diagnosis of pathogens for which biochemical methods for identification of species are known to be inaccurate¹⁷⁶ or to more rapidly arrive at a diagnosis.¹⁷⁷ In general, genotypic methods of identifying bacteria are based on identification of unique sections of DNA that are compared to characteristics of known library strains. Several PCR-based methods of identifying bacteria have been developed,^172,178,179 and real-time PCR tests are widely available. One commercially available test is able to accurately detect the presence of bacterial DNA of up to 16 mastitis pathogens and the β-lactamase gene but requires the use of aseptically collected milk samples to limit the number of false-positive results.¹⁸⁰ In one study, use of this test resulted in identification of bacterial DNA of potential mastitis pathogens in 43% of culture-negative milk samples.¹⁸¹ However, research has indicated that teat skin and teat canal flora may be the source of many minor pathogens identified in milk samples using PCR testing.¹⁸²

The use of the commercially available reverse transcription PCR (RT-PCR) test to evaluate quality of bulk tank milk has also been investigated.^183-185 As expected, PCR testing is useful to detect the presence of obligate udder pathogens (such as S. agalactiae) in bulk milk, but a single PCR test of bulk tank milk is not reliable for diagnosis of mastitis pathogens.¹⁸³ Interpretation of PCR test results of bulk tank milk for bac­teria that have possible environmental origins in bulk milk is difficult and not always more sensitive than bacteriologic culture.¹⁸⁵ PCR test results are interpreted relative to the cycling threshold (Ct) values, which indicate the number of cycles required to reach a signaling threshold. In general, the lower the Ct value, the greater the amount of the specified DNA in the sample, but similar to BTC results, PCR tests are not accurate to estimate the prevalence of infected cows within a herd.¹⁸³

Decision making for management of milk quality based on PCR results is complicated because DNA from both live and dead bacteria is detected. Just as the occurrence of a few colonies of environmental pathogens on a blood agar plate would not be sufficient evidence to indicate that a treatment is indicated, identification of bacterial DNA from milk is not always sufficient evidence on which to base a treatment protocol. To improve the utility of diagnostic methods based on DNA, aseptic samples should be collected¹⁸⁰ and the medical history and SCC of the cow should be reviewed before making treatment or culling decisions. For some pathogens, such as Mycoplasma spp., S. agalactiae, or S. aureus, the cow history will generally support the presumed diagnosis; for other pathogens the relationship may be less apparent. It is clear that the cost of using molecular methods will decrease, and therefore the use of these methods will increase. However, continued research is needed to help define practical ways of best using these methodologies.

Milk was long considered sterile, but researchers have been exploring the potential presence of a microbiota in milk from apparently healthy mammary quarters.^186,187 The presence of a 188190 commensal microbiota in the teat canal is well accepted,^188-190 but the mammary gland itself has long been considered to be a sterile environment.¹⁹¹ Based on a limited number of studies, some researchers¹⁹² have hypothesized that the bovine milk microbiota is composed of a complex and diverse community that includes both culturable and nonculturable organisms that are linked to intestinal flora and contribute to homeostasis and mammary gland health. Others have challenged this view,¹⁹¹ describing anatomic, immunologic, and practical research suggesting that while there is considerable evidence for the existence of a teat apex microbiota, the concept of a mammary gland microbiota is incompatible with known mammary gland immunobiology and warrants critical examination. This subject is an area of very active research, and clinical applications of these techniques in udder health programs have not yet been developed.

Epidemiology of Mastitis Pathogens

TRANSMISSION AND RESERVOIRS. Mastitis is an infectious disease that occurs when udders become infected with patho­gens, most of which are bacteria. With the exception of infections caused by a few unique microorganisms (such as Mycoplasma spp.), exposure of the teat sphincter to bacteria is the primary route of almost all mammary gland infections. Microorganisms that can potentially cause mastitis are typically categorized as either environmental or contagious based on the likely reservoir on the farm and the most probable source of the initial exposure.

Opportunistic organisms from the cow's surroundings are generally considered to be primarily environmental pathogens. Transmission occurs when teats are exposed to bacteria found in environmental sources such as fecal material, bedding, soil, and contaminated water.⁵⁸ Contamination of teat skin with environmental pathogens increases the probability of IMI, especially when defense mechanisms (such as the patency of the teat sphincter) are compromised. IMI with environmental pathogens can also occur when teat ends are not sufficiently disinfected before infusing antibiotics or when a contaminated infusion preparation or cannula is used. ^, Gram-negative bacteria, such as E. coli and Klebsiella spp., are the most com­monly isolated environmental pathogens.⁶ However, many gram-positive organisms, such as S. uberis and S. dysgalactiae can be found in the cows surroundings, and the initial infection with these pathogens is often from an environmental source. Although coliforms and streptococci are commonly isolated, many other organisms are found in the environment of cows and are capable of causing mastitis. The diversity of environ­mental mastitis pathogens is increasing as management of dairy farms intensifies and as a variety of bedding sources are used.^58,194 Reducing exposure to environmental pathogens is based on improving hygiene of cows, housing areas, and the milking process.¹⁹⁵

Mammary glands with chronic subclinical infections are the primary reservoir of contagious mastitis pathogens. Transmission occurs when teats are exposed to bacteria found in milk that came from an infected gland. In many instances, exposure to infected milk occurs during the milking process. Infected milk droplets can be found in teat cup liners, hands of milking technicians, towels used to wash or dry teats, or milk droplets that leak onto bedding surfaces. The presence of these organisms on teat skin increases the probability of infection. In some cases, abrupt vacuum fluctuations within the milking cluster (reverse pressure gradients) can propel contagious pathogens from the milk of an infected gland into the teat canal of a healthy gland.¹⁹⁶ The most common con­tagious mastitis pathogens are host-adapted bacteria that cause long-term subclinical infections. Historically, S. agalactiae was considered to be one of the most important causes of contagious mastitis¹⁹⁷; however, this obligate udder pathogen is controlled in most developed dairy regions and is not commonly isolated from milk samples of cows located in developed regions.^115,198,199 In recent years, S. aureus, and M. bovis have been the most prevalent contagious mastitis pathogens found in U.S. dairy herds.²⁰⁰ It is important to recognize that most organisms that establish chronic subclinical IMI can be transmitted in a contagious manner. Successful control programs for contagious pathogens are based on reducing the prevalence of subclinically infected cows, thus reducing the probability that healthy cows are exposed to milk that originated from infected udders.

The classification of organisms as contagious or environ­mental is a broad characterization of the most common sources of initial exposure to pathogens and does not preclude other potential routes of transmission¹⁴⁶ (see Table 36.2). Histori­cally, contagious pathogens were considered to be caused by highly contagious identical clones that were transmitted only among cows, whereas environmental bacteria were often considered to consist of diverse strains that were found only in the environment.²⁰¹ However, use of DNA fingerprinting has demonstrated that it is not uncommon to find several different strains of contagious organisms such as S. aureus occurring in cows on a single farm.²⁰² Similarly, a single dominant strain of Klebsiella spp. isolated from multiple cases of mastitis has been reported as evidence of contagious transmission of a supposedly environmental pathogen.²⁰³ The use of molecular methods has demonstrated that microbes are continually evolving in response to control measures and changes in farm manage­ment. While characterization of pathogens as “contagious” or “environmental” still provides a reasonable basis for initiating control programs, veterinarians should be aware of the pos­sibilities of additional routes of transmission and the need for comprehensive control programs that may exceed the typical recommendations.

PREVALENCE OF MASTITIS PATHOGENS. More than 130 organisms, including bacteria and some algae, yeasts, and fungi, have been documented to cause mastitis.^5,204 Although some regional differences in prevalence have been demonstrated, the distribution of pathogens that cause clinical and subclinical mastitis on modern dairy farms is remarkably consistent (see Table 36.1). In recent years, widespread adoption of standardized mastitis control procedures in developed dairy regions has resulted in a decreased prevalence of mastitis caused by con­tagious pathogens such as S. aureus and S. agalactiae (see Table 36.1).^115,142,143 Herds that fail to control these pathogens typically have failed to effectively implement well-known mastitis control procedures such as postmilking teat dipping and comprehensive use of antimicrobial dry cow therapy.¹⁹⁹ On most dairy farms, about 25% to 30% of milk samples obtained from cows secreting abnormal milk result in no significant growth of bacteria (see Table 36.1), usually because of an effective immune response to infection by opportunistic environmental pathogens. Coliform bacteria and environmental Streptococcus spp. are usually the most common pathogens identified from milk samples collected from cases of clinical mastitis on modern U.S. dairy farms (see Table 36.1).^6,28,205,206 In contrast, when milk samples are collected from cows with subclinical infections, few coliform bacteria are identified, indicating the short duration and clinical nature of most of these infections.^116,207-210 In regions that have controlled S. aureus and S. agalactiae, NAS are often the most prevalent pathogen group associated with subclinical infections (see Table 36.1). While epidemiological studies of mastitis pathogens within regions have resulted in consistent recovery of typical groups of pathogens, the distribution of pathogens often varies among farms based on individual risk factors. The implementation of effective control programs and correct treatments is based on understanding the etiology of mastitis, and sufficient diagnostic testing should be used routinely by veterinary practitioners who are working to control mastitis.

CHARACTERISTICS OF PATHOGENS. Pathogenesis, virulence, and prognosis of IMI are each influenced by important char­acteristics that vary among pathogens. General characteristics of the most common mastitis pathogens are described in Table 36.2. Depending on specific virulence factors, organisms infect different locations in the mammary gland, have differing abilities to cause systemic symptoms, vary in the expected duration of subclinical phases of infection, and differ in the expected rate of spontaneous bacteriologic cure (see Table 36.2). Understand­ing these differences is fundamental to the development of effective control programs.

MASTITIS CAUSED BY STAPHYLOCOCCUS SPECIES

Staphylococcus aureus. The genus Staphylococcus contains at least 50 species, and initial differentiation among species is based on their ability to coagulate plasma.⁵ Historically, staphylococci that do not coagulate plasma have been referred to as coagulase-negative staphylococci (CNS), but non-aureus staphylococci (NAS) is now the preferred terminology, as the group includes some species that coagulate plasma. Among staphylococci associated with bovine mastitis, S. aureus and Staphylococcus hyicus are coagulase positive, but S. hyicus is usually considered to be a minor mastitis pathogen. S. aureus is considered a major pathogen because of its impact on SCC and widespread prevalence. S. aureus has been isolated from bulk tank milk of about 40% of U.S. dairy herds,²⁰⁰ and about two thirds of producers who culture milk samples have reported its isolation,²¹ but the prevalence and economic impact vary tremendously among herds. Most IMIs caused by S. aureus are chronic and subclinical, with periodic bouts of mild clinical mastitis. However, clinical mastitis caused by S. aureus can result in systemic signs, or the affected mammary gland may become gangrenous. Herds with a large proportion of cows with mastitis caused by S. aureus tend to have higher BTSCCs and many cows with chronically increased SCCs (see Table 36.2).

IMI caused by S. aureus is diagnosed by isolating coagulase­positive staphylococci from milk samples of cows. When grown on appropriate media, colonies usually demonstrate hemolysis and other typical phenotypic characteristics.⁵ Occasionally some S. aureus strains are coagulase negative, thus definitive identifica­tion requires additional diagnostic testing. There are many different strains of S. aureus; some herds may have a predominant strain, whereas other herds may harbor diverse strains.^202,211 Some researchers have reported that variation among strains of S. aureus results in differences in transmission, virulence, and antibiotic susceptibility,^212,213 whereas others have reported similar outcomes independent of strain.^214,215

Although much is known about S. aureus, considerable knowledge gaps about pathogenesis still exist.^216,217 Some strains of S. aureus can adhere to and invade mammary epithelial cells and interstitial tissue, ’ ’ but variation in invasive potential among strains has been noted.²²⁰ A variety of mechanisms to avoid the immune response have been noted for S. aureus. The organism can resist phagocytosis by neutrophils with antiphago­cytic mechanisms such as a capsule that inhibits opsonization and a surface protein (protein A) that binds the Fc portion of host Ig. Leukotoxin production enables some S. aureus strains to kill phagocytes²²¹ while other mechanisms allow S. aureus to survive within leukocytes and epithelial cells and induce apoptosis.²¹⁹ Some strains of S. aureus produce enzymes and exotoxins that damage mammary cells and result in fibrosis and abscess formation.²²² Many strains produce β-lactamase, convey­ing resistance to antibiotics such as penicillin and amoxicillin. Others convert to L-form or small colony variants, which can persist even after antibiotic therapy.^215,223 Certain S. aureus strains form biofilms, making them inaccessible to phagocytes and antibiotics,^224-226 and some but not all biofilm-associated proteins have been associated with chronic infections.^220,227 For these reasons, S. aureus IMIs seldom resolve spontaneously and usually respond poorly to antimicrobial therapy.²²³

Farmers who purchase replacement heifers or lactating cows without screening the animals or source herds often have herds with greater prevalence of S. aureus as compared to closed herds, illustrating the importance of biosecurity in mastitis control. The prevalence of infected cows within a herd is an important risk factor for new infections. Cows with chronic S. aureus infections or with characteristics that predict a low likelihood of cure should be culled as soon as economically feasible or segregated and milked last. Alternatives are simply to stop milking the infected gland or to induce cessation of lactation in an individual gland.

Non-aureus Staphylococci (Previously Referred to as Coagulase-Negative Staphylococci). In most studies, NAS are the most prevalent bacteria isolated from milk samples obtained from cows with subclinical infections and from the mammary secretions of dry cows and prepartum heifers (see Table 36.1). Based on use of traditional microbiological methods of identification, there are at least 50 species of NAS, and many have been isolated from IMIs of dairy cows.^5,228,229 Epidemiological differences among various species have not been well defined. While researchers have demonstrated some variation in SCCs and persistency of IMI among NAS species, these differences have not been shown to affect milk yield,^230,231 and control programs are generally nonspecific. Historically, a thorough understanding of epidemiologic characteristics and differences in pathogenesis among NAS species has been limited by the relatively poor discriminatory ability of phenotypic or biochemical diagnostic tests.²³² The accuracy of identification of NAS associated with bovine mastitis has been improved by the use of genotypic methods (such as sequencing), and the use of MALDI-TOF MS by diagnostic laboratories has allowed 159233

for improved discrimination among species.¹’

Most recent surveys using modern discriminatory methods indicate that Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans are the most prevalent species associated with bovine mastitis, but 231234235

variation among studies has been noted. ’⁴’ Staphylococci are the most common organisms recovered from mastitis in heifers, and a variety of NAS species have been recovered from teat skin, the streak canal, and precalving udder secretion obtained from heifers.^21,236

IMI caused by NAS typically results in subclinical infection that is associated with only modest increases in SCC²³⁷; thus despite the widespread prevalence, NAS are often referred to as “minor” mastitis pathogens.²²⁹ Most infections caused by NAS remain subclinical, with SCCs lower than 500,000 cells/mL. However, when NAS infections persist they may be character­ized by greater increases in SCCs.²³⁸

Although NAS account for about 15% to 20% of subclinical IMI, they are recovered from a relatively small proportion of cases of clinical mastitis, and clinical signs are usually mild (Table 36.1).^146,239 The rate of spontaneous cure is often high,²⁴⁰ but intramammary treatment used for cases of clinical mastitis caused by NAS usually results in bacteriologic cure rates that exceed 80%.^229,237 The use of β-lactam-based intramammary antibiotics at dry-off is often successful in treating subclinical IMI caused by NAS. Treatment of subclinical IMI caused by NAS during lactation is unlikely to be profitable because a potential increase in milk yield after treatment has not been documented. There are no specific control measures for NAS mastitis. Effective premilking and postmilking teat dipping, comprehensive antibiotic treatment of cows at dry-off, and attention to udder and milking hygiene are recommended.

MASTITIS CAUSED BY STREPTOCOCCUS SPECIES

Streptococcus agalactiae. S. agalactiae is generally consid­ered to be an obligate udder pathogen, and control measures that focus on reducing the prevalence of infected cows have been highly effective.²⁴¹ Historically, IMI caused by S. agalactiae was the most common cause of bovine subclinical mastitis, and most preventive practices used on modern dairy farms were developed to aid in reducing transmission of this highly contagious organism.¹⁹⁷ On modern North American dairy farms, control of S. agalactiae has been successful, and this organism is rarely isolated from individual cow milk samples (see Table 36.1) or bulk tank milk obtained from U.S.²⁰⁰ or Canadian dairy farms.¹⁴³ However, without proper attention to biosecurity and milking procedures, S. agalactiae can be introduced and spread rapidly through a herd, resulting in substantial economic loss. Mastitis caused by S. agalactiae is usually subclinical and if not treated can persist for months or years (see Table 36.2). Subclinical infection may be interspersed with bouts of mild clinical mastitis, but only rarely will cows exhibit systemic signs.

S. agalactiae adheres to epithelial cells in the mammary gland and localizes primarily in the ducts. Milk production declines when ducts become blocked with cells and debris and the associated alveoli involute. Early treatment can result in increased milk production because the superficial nature of the infection results in minimal damage to the milk secretory cells. However, tissue damage, fibrosis, and a permanent decrease in milk production can occur if infection becomes chronic. S. agalactiae-infected glands tend to shed high con­centrations of bacteria and somatic cells in milk. and this organism should be suspected in the rare instances when a BTSCC exceeds regulatory limits.²⁴

S. agalactiae is easily isolated from milk samples and is diagnosed when esculin-negative, CAMP-positive streptococci are identified in milk samples obtained from bulk tanks or from individual cows. S. agalactiae is usually an obligate udder pathogen so Rl-PCR is useful for rapid diagnosis of infected cows.¹⁷⁶ In some regions that have not successfully controlled this organism, there is evidence of adaptation to environmental niches²⁴²; however, this is not common. When the organism is found in bulk milk or milk samples of affected cows, a sampling plan should be developed to screen the herd to identify and treat all infected cows. Antibiotic treatment has formed the basis for control of S. agalactiae, and intramammary antibiot­ics have been found to be more efficacious as compared to intramuscular treatment using penethemate. In instances where use of antibiotics is not an option (e.g., organic dairy farms in the United States), it is especially important to identify and cull infected cows. Control programs for S. agalactiae are based on eradication through identification, treatment, culling, and biosecurity.

Environmental Streptococcus Species. A number of streptococci and closely related organisms are frequently isolated from milk samples collected from cows with clinical and subclinical mastitis (see Table 36.1). S. uberis and S. dysgalactiae are the most common streptococcal species recovered from IMI, but other species such as Streptococcusparauberis, Streptococ­cus equinus, Streptococcus Saccharolyticus, Streptococcus salivarius, and Streptococcus canis have also been associated with mastitis.²⁴⁴ In addition, there are a number of gram-positive, catalase­negative cocci such as Enterococcus faecalis, Enterococcus faecium, Aerococcus viridans, and several species of Lactococcus that have similar reservoirs and are often grouped with streptococci.²⁴⁵ These streptococcal-like organisms are collectively referred to as environmental streptococci because the primary exposure is often in cow housing areas. Streptococcal organisms can be cultured from many materials commonly used for bedding, including contaminated sand.¹⁹⁴ Many of the IMIs caused by environmental streptococci result in subclinical infections, and strains that become chronic may be transmissible among cows in a contagious manner.^246-248

Environmental streptococci are shed in the feces of cattle and are often ubiquitous in the dairy farm environment.²⁴⁹ Infected dogs and cats can carry S. canis in their respiratory and urogenital tracts and serve as potential sources of infection for 246 248 249

cows.²⁴⁶,²⁴⁸ S. ubens is found in the vagina of cows,²⁴⁹ but genital tract secretions probably play little role in the transmission of infection. Teats are often exposed to environmental streptococci that are present in bedding contaminated with feces¹⁹⁴; straw is well known to support growth of S. uberis. S. uberis is also well known to affect cows on pastures and is the most frequent cause of mastitis in New Zealand dairy herds.^250,251 High-traffic cow lanes and areas of pastures or lots where cows congregate and defecate harbor S. uberis and serve as reservoirs.²⁴⁹

Approximately 50% of IMI caused by environmental strep­tococci are initiated during the dry period.²⁵² The mammary gland is highly susceptible to environmental streptococcal infection during the early and late stages of the dry period, when mammary secretions accumulate and host defense mechanisms are compromised.^116,253,254 Risk factors for IMI caused by S. uberis are similar to those for IMI caused by S. aureus.²⁵⁵ IMI is accompanied by a rapid influx of neutrophils into the mammary gland and a resultant increase in SCC. Based on experimental inoculation studies with S. uberis, the recruited neutrophils are unable to fully prevent bacterial multiplication. Ineffective host response probably explains the prolonged infections and persistently high SCCs seen in some cases.

Although many IMIs caused by environmental streptococci spontaneously cure within 30 days, about one third of infections persist for long periods, sometimes longer than a lactation. Up to 50% of IMIs caused by environmental streptococci may result in clinical signs,²⁵² but only about 10% to 20% of those cases will become systemically ill.^6,256 Recurrent episodes of clinical mastitis are common.²⁵⁷ Control of IMI caused by environmental streptococci is based on reducing exposure in housing areas and ensuring that cows remain healthy, clean, and dry.

MASTITIS CAUSED BY COLIFORMS

Characteristics Shared Among Coliform Bacte­ria. Coliform bacteria are defined as gram-negative, rod-shaped organisms that can ferment lactose.⁵ These organisms are fairly easy to identify because growth on MacConkey agar results in a characteristic pink colony. E. coli and several other genera such as Klebsiella, Enterobacter, and Serratia (some of which are non-lactose fermenting) are commonly associated with bovine mastitis (see Table 36.1), whereas other coliforms such as Citrobacter spp. only rarely cause mastitis. Coliform organisms include both pathogenic and nonpathogenic strains that are ubiquitous in the dairy farm environment and frequently shed in feces. Within individual bacterial species, a wide variety of strains are capable of causing mastitis.²⁵⁸ Teats are initially exposed to these organisms in housing or pasture areas, thus coliforms are considered to be primary environmental patho­gens. Organic bedding materials, such as straw, wood chips, sawdust, recycled manure or solids from methane digesters, pelleted corncobs, and newspaper, support the growth of coliform bacteria.²⁵⁹ Environmental conditions such as increased ambient temperatures and increased rainfall influence the growth of bacteria and thus the extent of teat end exposure and risk of intramammary infection. Large numbers of gram-negative organisms are present in bedding that is based on use of manure.^194,208 As compared to sand bedding, manure-based bedding materials have been associated with increased clinical and subclinical mastitis and reduced milk yield.²⁶⁰ Likewise, management decisions such as increased housing density, poor stall design, failure to use sufficient bedding materials, or poor pasture management can increase the probability that teats will be exposed to coliform bacteria in manure.

In early lactation, clinical mastitis caused by coliforms may be the result of IMI acquired during the dry period.^116,261,262 However, coliform infections can be acquired at any time during lactation, particularly if the exposure is great and primary defense mechanisms, such as the patency of the teat sphincter, are compromised.

After coliform bacteria infect the mammary gland, they multiply rapidly but most do not adhere to or invade the epithelial cells.²⁶³ If the cow’s immune response is rapid and efficient, infection will be quickly eliminated and there will be little long-term impact on cow health or productivity. Although the majority of clinical cases of mastitis caused by coliform bacteria are mild, up to 30% of cases may result in systemic signs such as anorexia, fever, or a marked drop in milk production.^6,256 On most farms, IMI caused by coliform mastitis accounts for the majority of mastitis cases accompanied by systemic signs.²⁶⁴ Development of systemic signs and severe

disease are primarily associated with characteristics of the cow that influence its ability to respond to the infection.²⁶³ When influx of neutrophils is delayed or phagocytosis or intracellular killing mechanisms of neutrophils are impaired, bacterial multiplication continues, resulting in greater concentrations of inflammatory mediators and more severe clinical disease. Severe cases occur most frequently in the periparturient period and during early lactation.^72,265,266

The outcome of clinical mastitis caused by coliform bacteria depends on the severity of the case, which usually depends on the balance between the dose (magnitude of of exposure) and the ability of the cow to response immunologically. Increased or subnormal rectal temperature, reduced rumen contraction rate and amplitude, marked dehydration, and marked depression are associated with increased risk of death, culling, or poor return to milk production.^145,267 Severe neutropenia and high bacterial concentration in the milk¹⁴⁵ are also indicative of a poor prognosis. Cows in early lactation do not develop neu­tropenia to the extent observed in later lactation; therefore the leukogram is not a good indicator of mastitis severity in early lactation.²⁶⁸

In contrast to environmental streptococcal mastitis, intra­mammary antibiotic treatment at dry-off is considered to be less effective in preventing coliform infections during the dry period.^269,270 The spectrum of activity of many antibiotics (β-lactams and macrolides) used for dry cows in the United States is primarily directed toward gram-positive pathogens. Infusion of internal teat sealant at dry-off reduced the incidence of new E. coli infections compared with infusion of antibiot­ics (cephalonium) in one study.²⁷¹ However, the risks of new gram-negative IMIs during the dry period and of clinical mastitis during early lactation were similar when quarters were treated with teat sealant plus antibiotics (cloxacillin) or antibiotics alone.²⁷² The use of internal teat sealant combined with appropriate intramammary antibiotics has been shown to result in reduced frequency of new IMI caused by coliform bacteria.²⁷³

Control of mastitis caused by coliform bacteria is based on reducing teat end exposure to these organisms. This is especially true for periparturient cows that may have compromised immune responses. Improving the hygienic conditions of the cows' surroundings and during the milking routine, reducing cow density, and ensuring that cows have access to an adequate diet are fundamental steps used to reduce the incidence of coliform infections. Although coliforms are often referred to as a group, there are some important differences in pathogenicity and expected outcomes of IMI among the coliform genera.

Escherichia coli. Most IMI caused by E. coli has a short duration, and these organisms are rarely isolated from cases of subclinical mastitis.²⁷⁴ While many producers may believe that E. coli typically causes severe mastitis, research demonstrates that about one third of cases present with mild symptoms (only abnormal milk), one third present with moderate symptoms (abnormal milk accompanied by local signs in the udder), and one third present with systemic signs.^6,256 Most cases of clinical mastitis caused by E. coli are preceded by low SCCs, and the transient nature of most E. coli infection is evidenced by the rapid return of SCCs to relatively normal levels within weeks of occurrence of a clinical case.²⁷⁵ Although some researchers have proposed that specific strains of E. coli are more pathogenic in cows,²⁷⁶ genomic studies indicate that virulence traits are highly diverse among strains^277,278 and E. coli is considered to be a facultative opportunistic pathogen that typically originates from the gastrointestinal tract.²⁷⁷ However, about 5% of E. coli cases can result in persistent infections that may lead to 279280 recurrent clinical mastitis in the same or another quarter., Differences in gene expression between E. coli strains that cause transient versus persistent infection have been identified, and transient strains have been reported to be more sensitive to complement-mediated killing as compared to persistent strains, suggesting that both host and pathogen factors may influence response to IMI.²⁸¹

Klebsiella Species. Klebsiella oxytoca and Klebsiella pneu­moniae are the two most common species associated with bovine mastitis, and molecular studies have indicated that some organisms previously identified as Klebsiella spp. are actually Raoultella spp.²⁸² The clinical presentation and management of mastitis caused by Raoultella spp. are identical to recom­mendations for control of mastitis caused by Klebsiella spp.²⁸² Although the distribution of severity of symptoms is similar for clinical mastitis caused by both Klebsiella and E. coli,²⁸³ the impact of Klebsiella mastitis on milk yield is significant and long lasting.^28,205 In contrast to IMI caused by E. coli, IMIs caused by Klebsiella spp. are often of much longer duration and accompanied by long duration of increased SCCs.²⁸² While the origin of initial IMI may be environmental, between-cow transmission can occur during periods of subclinical infection. Sawdust bedding has been implicated in outbreaks of Klebsiella mastitis, but Klebsiella species can proliferate rapidly in other bedding materials, and fecal shedding from healthy dairy cows has been documented.²⁸⁴

Serratia Species. Serratia marcescens and Serratia liquefaciens are the species that are most commonly associated with bovine mastitis. Many Serratia species produce bright-red colonies on blood agar, but they are easily outcompeted by other pathogens and prevalence may be underestimated unless special media and cultural conditions (e.g., incubation at 20° C) are _used.285,286

In contrast to mastitis caused by other coliforms, IMI caused by Serratia spp. often persists in a subclinical form for many months. Field observations and experimental infections have demonstrated that Serratia spp. are less pathogenic than most other coliform infections, probably because they stimulate less 287

of an immune response.²⁸⁷ In contrast to mastitis caused by other coliform bacteria, few cases of mastitis caused by Serratia result in severe clinical signs.^288,289 However, Serratia infection persists longer than E. coli infection. A mean duration of 55 276288

days has been reported,²'⁶,²⁸⁸ but many infections persist for 6 to 10 months.^285,289 Substantial SCC increases in cows and bulk milk are often associated with herd outbreaks of mastitis caused by Serratia spp.²⁸9,²⁹⁰

Serratia IMI can originate during lactation or the dry period. In one longitudinal study, 48% of Serratia infections were acquired during the first half of the dry period and 31% during the second half, with only 21% initiated during lactation.²⁸⁸ IMI caused by Serratia can originate from environmental reservoirs such as organic bedding material.^285,286 However, mastitis caused by Serratia is frequency associated with con­taminated chlorhexidine teat dips or teat dip cups.^282,291 In one outbreak that used molecular analysis to identify a source, the diversity of Serratia strains identified indicated that the chlorhexidine teat dips became contaminated with unique strains 270 282 276 277

at each farm.²'⁰,²⁸² Antibiotic therapy is usually ineffective,²'⁶,²'' but up to 50% of infected quarters may eventually self-cure.^288,289 Segregation of chronically infected cows may help to contain an outbreak.²⁹⁰ Culling is recommended for cows with recurrent cases of clinical mastitis or persistently high SCCs.

MASTITIS CAUSED BY OTHER ORGANISMS

Mycoplasma Species. Mycoplasma spp. are well known pathogens of dairy cows that infect a number of organs and cause a variety of cattle diseases.²⁹² Mycoplasma spp. are associated with otitis media in calves, infections of the urogenital tract, arthritis in calves and cows, pneumonia, and mastitis. Infections caused by Mycoplasma spp. can be recognized in disease out­breaks that present with a range of clinical signs, including death of adult cows,²⁹³ and often result in significant economic losses for dairy farms.²⁹⁴ Mycoplasma has been isolated from the bulk tank milk of about 9.4% of U.S. dairy herds, but the prevalence is strongly associated with herd size and region. In 2014, the USDA⅛ National Animal Health Monitoring System (NAHMS) reported that Mycoplasma was isolated from 25% of bulk tank milk samples obtained from herds that contained more than 500 cows, in contrast to 6% and 0% of herds containing 100 to 499 cows and 30 to 99 cows, respectively.²¹ Risk factors for Mycoplasma mastitis include commingling of cattle from multiple herds, off-site rearing of calves, and stressors such as overcrowding, nonhygienic milking practices, and insufficient biosecurity programs.

Although a number of Mycoplasma spp. are considered pathogenic for cattle, the most common species associated with mastitis include M. bovis, Mycoplasma californicum, and Mycoplasma bovigenitalum?⁹² M. bovis is the most common species associated with bovine mastitis. Mycoplasma species can be isolated from a variety of mucosal surfaces of both normal and ill cattle. However, there is increasing evidence that M. bovis is an important cause of chronic nonresponsive pneumonia in cattle.²⁹⁵ While Mycoplasma spp. can be isolated from the nares of clinically normal cattle, several researchers have noted that the prevalence of nasal shedding is greater in herds that have problems with Mycoplasma mastitis as compared to herds that have not identified a Mycoplasma mastitis problem.²⁹² At the individual animal level, neither nasal nor vaginal swabs are consistently positive in clinically affected animals,²⁹⁶ and use of serology to diagnose individual animals has not been shown to be sufficiently predictive to be used in control programs.²⁹⁷ Colonized cattle may remain asymptomatic or develop bron­chopneumonia, otitis, polyarthritis, or mastitis. Because of the unique nature of carriage and transmission of this organism, veterinarians should review detection, diagnosis, and treatment of bovine respiratory disease as part of an investigation into mastitis outbreaks associated with Mycoplasma spp. In dairy herds with Mycoplasma mastitis, otitis media and otitis interna, respiratory disease, or swollen joints may be seen in calves, especially if unpasteurized milk is ingested.^298,299

Transmission of Mycoplasma spp. is unique in that multiple routes of infection of the mammary gland are possible. Asymptomatic carrier animals may infect herdmates by shedding the organisms in nasal or vaginal secretions, feces, or milk,²⁹² and infected semen can serve as a route of infection in naive herds.³⁰⁰ Infection can occur at milking time when teats are exposed to fomites infected with Mycoplasma organisms that originated from the udder of another cow. However, IMIs can also occur when Mycoplasma spp. are spread from other infected body sites such as the respiratory tract. In one study, intramam­mary inoculation of Mycoplasma species into a single mammary gland was followed by detection of phenotypically identical Mycoplasma species in extramammary body sites, blood, and noninoculated mammary glands.^301,302 Biddle and colleagues³⁰³ found that isolates of Mycoplasma species from the milk, mammary gland parenchyma, and supramammary lymph nodes of infected cows all had the same pulse-field gel electrophoresis pattern and that all cows had at least one extramammary isolate with the same pattern. These findings suggest that cows with Mycoplasma mastitis frequently have disseminated infections and that internal transmission may occur.

The presentation of mastitis outbreaks caused by Mycoplasma spp. is extremely variable among farms. On many farms, most IMIs caused by Mycoplasma result in persistent subclinical infec­tions. However, clinical mastitis often affects multiple glands (as a result of internal transmission of infection) and will not respond to antibiotic therapy. Many outbreaks of disease caused by Mycoplasma are not recognized because diagnosis depends on the use of appropriate laboratory methods. Mycoplasma spp. have unique growth requirements and must be cultured using appropriate media and incubation under specific conditions such as 10% carbon dioxide.³⁰⁴ Prolonged freezing of milk samples reduces the concentration of viable organisms^153,305 and may result in false-negative diagnoses, but broth enrich­ment before culturing can enhance detection of Mycoplasma species.¹⁵³ When culture is used for diagnosis, the plates must be allowed to incubate for up to 7 to 10 days, as some colonies will grow very slowly. The identification of Mycoplasma DNA in milk is indicative of IMI, so PCR testing is useful because a diagnosis can be achieved more rapidly.¹⁷² PCR-based diagnostic methods have acceptable limits of detection (samples can then be collected and pooled to identify individuals for further testing. Cows with clinical mastitis and those with high SCCs are good candidates for testing, but some infected animals may not exhibit clinical mastitis and may maintain relatively low SCC values, so diagnostic testing should continue until the bulk tank tests negative and the symptoms of an outbreak subside.

Mycoplasma organisms lack a cell wall, thus most antibiotics approved for use in dairy cattle are not effective. Antibiotic treatment during lactation or at dry-off is not useful for control of Mycoplasma mastitis. Mycoplasma spp. have the ability to inhibit the inmate immune response³⁰⁶ and enhance neutrophil apoptosis,³⁰⁷ thus enhancing dissemination and reducing the probability of spontaneous cure. Although some IMIs may resolve spontaneously, infected cows should be considered permanently infected. Even if repeated culturing of milk from an individual quarter yields negative results, infection might persist in an extramammary site and spread to the mammary gland. For this reason, the most common and least risky control strategy is based on an aggressive testing and culling program for all cows diagnosed with Mycoplasma mastitis.³⁰⁸ However, even aggressive diagnosis and culling programs may not prevent periodic Mycoplasma mastitis incidents if infections persist asymptomatically in extramammary sites. Some herds are able to control Mycoplasma mastitis without aggressive culling by implementing within-herd segregation, effective biosecurity, and excellent milking techniques. This strategy is more risky as even with these practices, airborne transmission among cows may still occur. Vaccination against M. bovis is possible, as bacterins are available, but the efficacy of these vaccines for control of mastitis has not been demonstrated. Vaccination against M. bovis is hindered because of variable expression of a diverse and ever-changing set of genes encoding variable surface lipoproteins (Vsps).³⁰⁹

Overall, control of M. bovis is based on identification of infected cows, prevention of transmission by excellent biosecu­rity, and implementation of milking and management programs that prevent new infections. As with other pathogens that can be transmitted in a contagious manner, effective postmilking teat dipping is required. Mycoplasma species are susceptible to most germicides found in teat dips.³¹⁰ Effective premilking and postmilking teat dipping and other measures used to prevent the spread of contagious pathogens in the milking parlor help to limit Mycoplasma mastitis transmission. Care must be taken to ensure that hospital pens do not become a source of transmis­sion either through direct contact with infected cows or through fomites or airborne transmission.³¹¹ Control of respiratory disease and provision of housing with excellent ventilation is also necessary. Pasteurization of waste milk reduces the risk of Mycoplasma transmission to milk-fed calves.²⁹⁹

Pasteurella multocida. Pasteurella multocida is found in the upper respiratory tract of cattle and can be considered both normal flora and a contributor to bovine respiratory disease complex. This organism is occasionally associated with bovine mastitis. Most episodes are sporadic, but herd outbreaks can occur.³¹² Mastitis caused by P multocida can be recognized by increased incidence of clinical mastitis and by an increase in BTSCCs that is indicative of cows with subclinical infections. Hematogenous or lymphatic spread of P multocida from the respiratory tract to the mammary gland has been proposed, and P. multocida has been isolated from the blood of cows with severe coliform mastitis.³¹³ Contamination of teats by nasal secretions (e.g., from nursing calves or intersucking cows), cow-to-cow spread at milking time, or inappropriate infusion practices may contribute to outbreaks. Antibiotic treatment has histori­cally been unrewarding, and infected cows are often culled. However, spontaneous resolution appears to occur in some cases. Control of herd outbreaks associated with P. multocida should be directed at reducing the possibility of cow-to-cow transmission at milking time and improving ventilation of cow housing areas.

Prototheca Species. Prototheca species are unicellular algae that are widespread in nature and occur throughout the world in habitats that contain moisture and organic matter.⁵⁷ Prototheca spp. have been isolated frequently from feces of cows and other warm-blooded animals such as pigs and rodents.^314,315 On dairy farms, several different Prototheca species have been found in moist areas such as soil, mud, vegetation, and water.³¹⁴ Although Prototheca blaschkeae and Prototheca zopfii genotypes 1 and 2 have been associated with bovine mastitis,^316,317 P zopfii genotype 2 is the main species associated with herd outbreaks of Prototheca-related mastitis.^318,319 Initial IMI is likely due to environmental reservoirs, but several risk factors for Prototheca mastitis have been identi- fied.⁵⁹ Important risk factors include the use of intramammary infusions of nonapproved products and the use of internal teat sealants at dry-off. On many farms, Prototheca is ubiquitous in the environment, and these risk factors illustrate the importance of hygienic technique when performing intramammary infusions. When Prototheca is identified as a cause of mastitis, veterinarians should investigate treatment practices and ensure that teats are thoroughly cleaned before infusion and that intramammary prod­ucts are not warmed by being placed in potentially contaminated farm water before infusion into the udder.

P zopfii will grow on blood agar and can be detected by microscopic examination of Gram-stained colonies; however, the colonies are easily overgrown and may be underdiagnosed. Diagnosis can be improved by culturing milk on selective media. Prototheca isolation medium (PIM) contains inhibitory substances that facilitate identification of the organism and is often used in mastitis laboratories. Identification of specific genotypes is not routinely performed but can be done for research or epidemiologic purposes using several molecular techniques.³¹⁸,³²⁰

IMI with pathogenic Prototheca can result in both subclinical and clinical symptoms, and the infections can occur sporadically, endemically, or acutely. Most clinical cases are of mild or moderate severity. Infected cows may become chronically infected and experience persistently increased SCCs. Shedding of P. zopfii in milk can be persistent or intermittent.³²¹ Most Prototheca IMIs are probably acquired from the environment, but the possibility of transmission among cows in a contagious fashion should be recognized and prevented as part of a control program.

Treatment of mastitis caused by Prototheca is not recom­mended. P zopfii is resistant to antimicrobial therapy, and infection can persist through the dry period.¹⁹⁸ Therefore identification, segregation, and progressive culling of infected animals are required to control Prototheca mastitis. On most farms, searching for point sources of environmental contamina­tion will not be useful; however, hygienic conditions in the environment should be improved. Culling of infected cows should be coupled with identification and avoidance of con­taminated environmental sites, enhanced cleaning of feed bunks and watering devices, and use of milking procedures that ensure clean teats and minimize contagious spread of infection.

Corynebacterium bovis. Corynebacterium bovis is commonly found in the streak canal and can cause occasional IMI that results in mild increases in SCC and very little impact on milk yield.³²² It is rarely a cause of clinical mastitis. C. bovis can be cultured on routine media such as blood agar, but colonies may not be apparent until after 48 hours of incubation. C. bovis can be a frequent contaminant in milk samples, and DNA from this organism is frequently identified in association with other bacteria when PCR testing is used on milk samples.¹⁷⁸ This organism can be transmitted from cow to cow at milking time, but the use of premilking and postmilking teat germicides is effective in controlling transmission.

Trueperella pyogenes. Trueperella pyogenes is the current name assigned to an organism that previously was named Arcanobacterium pyogenes (formerly Actinomyces pyogenes or Corynebacterium pyogenes). IMI with this organism usually results in acute purulent symptoms of clinical mastitis. In most instances, treatments are not effective, and mastitis caused by this organism has a poor prognosis.⁵ Few cases remain subclinical, and the identification of this organism in bulk tank milk usually results from failure to detect and divert milk from cows with clinical symptoms. The outcome of most cases occurring in lactating cows is loss of the quarter or culling of the cow.³²³ Transmission among cows can occur by flies or through environmental exposure of teat skin, especially if teat ends are damaged.

Historically, this organism was considered one of the etio­logic agents of “summer mastitis,” a condition of prepartum heifers and nonlactating cows that has been recognized mainly in Europe and Japan during summer months.³²⁴ In most cases, anaerobic bacteria (e.g., Peptococcus indolicus, Bacteroides spp., Fusobacterium necrophorum) and facultative anaerobes (e.g., S. dysgalactiae') are also involved.^324,325 Summer mastitis is character­ized by a swollen, hard, painful mammary gland containing purulent, foul-smelling secretion. Acutely affected animals typically demonstrated systemic signs ranging from lethargy and reduced feed intake to high fever, severe depression, abortion, and death. Systemic administration of procaine penicillin G often resolves systemic illness, but neither systemic nor intra­mammary antibiotics are effective at eliminating IMI.³²⁵ The outcome of most episodes of summer mastitis is chronic clinical mastitis or a nonfunctional quarter. Horn flies are believed to be responsible for transmitting summer mastitis. Because T. pyogenes is often found in the environment on farms and on body sites of cattle, teat skin contamination may predispose to infection, as occurs with coliform or environmental streptococcal mastitis, particularly if teat defenses are compromised.

Control of summer mastitis is accomplished by fly control, infusion of mammary glands with antibiotics at dry-off, appropriate environmental hygiene practices and stocking density, prevention and treatment of teat lesions, and prompt recognition and segregation of infected cows. Amputation of the teat, which facilitates drainage of secretions, increases environmental contamination and should not be done unless the cow can be segregated.

Yeast Mastitis. Yeast are microorganisms that are ubiquitous in the environment of dairy farms, and they can cause opportunistic IMIs. Sources of exposure of teats include contact with soil, plants, and water and feedstuffs such as total mixed rations.³²⁶ Candida species, Tricbosporon beigelii, and other yeast organisms are associated with mastitis in individual cows or herds.⁵ Yeast infection usually follows intramammary antibiotic treatment or teat skin injury. Outbreaks often occur when homemade infusion products or associated equipment (e.g., needles, syringes, bottles, cannulas) are contaminated or when the teat is not adequately disinfected before infusion., In one study, six cows treated with a homemade intramammary antibiotic solution contaminated with T. beigelii all developed fever, hypogalactia, and swollen mammary glands within 2 weeks, and two of the six died. When the producer continued to use homemade solutions, an additional 23 cows (over half the herd) became infected, and the herd was dispersed.³²⁷ Yeast have been cultured from towels and teat cup liners used on infected cows,

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suggesting that contagious transmission may occur.³,

On blood agar, yeast colonies appear similar to NAS colonies, so it is important to examine the colonies microscopically. Sabouraud dextrose agar with antibiotics is a common alternative medium that selects for yeasts. Yeast infections are not sus­ceptible to antibiotic treatment, and infusion of antibiotics may potentiate infection. The use of antifungal agents is not allowed in the United States, but experimental use has not been demonstrated to be effective. Infection (especially with Candida species) often resolves spontaneously within 2 to 4 326329

weeks,³, but cows with severe or recurrent clinical mastitis, reduced milk production, or persistently high SCCs should be culled. Control of yeast mastitis includes aseptic intramam­mary infusion procedures; use of single-dose, commercially prepared antibiotic infusion products; segregation of infected cows at milking time; and use of individual rather than shared towels. Feeding contaminated silage or total mixed ration can promote fecal excretion of yeast and has been blamed for outbreaks of yeast mastitis in some instances.

Pseudomonas Species. Pseudomonas spp. are considered to be environmental pathogens that are commonly isolated from water sources on dairy farms.⁵ Pseudomonas spp. typically appear on blood agar as white or gray hemolytic colonies and may produce a grape-like odor. Pseudomonas aeruginosa is the most common species isolated from milk of mastitic cows. IMI with Pseudomonas spp. can result in severe clinical mastitis that may develop into gangrene.³³⁰ However, chronic subclinical mastitis with periodic bouts of mild clinical mastitis is the more common presentation.

After IMI with Pseudomonas spp. is established, it usually persists, and antibiotic therapy is unsuccessful. This is caused in part by production of factors that inhibit host defenses and reduce antibiotic efficacy, such as biofilm.⁷³ Cows with persis­tently high SCCs or repeated bouts of clinical mastitis should be culled, or individual glands may be dried off.³¹¹

IMI in cows usually occurs when teats come in contact with contaminated water, and the use of water to wash teats before milking has frequently been implicated in outbreaks of mastitis caused by Pseudomonas spp. Water sources should always be investigated when Pseudomonas spp. are responsible for outbreaks of mastitis on dairy farms. Predipping for teat disinfection rather than washing with water reduces the risk of infections caused by contaminated water sources. Similar to IMI caused by Prototbeca, mastitis caused by Pseudomonas has been attributed to use of contaminated intramammary infusion products⁵⁶ or teat cannulas, inadequate teat end sanitation before

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drug infusion, contaminated teat wipes,³³¹ and exposure of cows to stagnant water. Investigation of a herd problem should focus on hygienic practices for intramammary infusion practices and reduction of exposure to water in the environmental and milking process.

Nocardia and Mycobacterium Species. Nocardia species and mastitis-causing Mycobacterium species are saprophytes and therefore may cause IMI when teats are exposed to unhygienic environmental conditions. However, both Nocardia spp. and Mycobacterium spp. are uncommon causes of mastitis that have typically been associated with unhygienic use of intramammary infusions or use of contaminated intramammary products.^5,332,333 Both of these organisms have been associated with sporadic cases or herd outbreaks of mastitis. Symptoms are variable and can range from greatly increased SCC³³⁴ to death. A tenta­tive diagnosis can be made by observing acid-fast rods or gram-positive rods with branching filaments in a milk smear, but culture of aseptically obtained milk samples is required to diagnose and differentiate Nocardia and Mycobacterium species. Prolonged freezing can reduce recovery of Nocardia from milk samples. Antibiotic therapy is futile. Nocardia is a potentially zoonotic pathogen, and affected animals should be segregated and culled. Control of mastitis caused by these organisms involves proper intramammary infusion practices and general environmental hygiene.