**2. Prevalence of mastitis in unbred and primigravid heifers**

A greater focus on heifer mastitis began in the mid 1980s after several dairy producers in Louisiana complained to university researchers that a large percentage of their heifers were

© 2012 Nickerson, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Nickerson, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

freshening with clinical mastitis. Subsequent study of breeding age animals in a research herd revealed that IMI may be diagnosed as early as 6 months of age, and infections persisted throughout pregnancy and into lactation (Boddie et al., 1987). Other studies demonstrated that greater than 90% of breeding age and bred heifers (12 - 24 months of age) may be infected (Trinidad et al., 1990b). Most of the infections were shown to be caused by the CNS (*Staphylococcus chromogenes* and *Staphylococcus hyicus*) followed by *Staph. aureus* (20%). Mixed isolates of CNS and *Streptococcus* species were also found.

The prevalence of mastitis in 10 unbred Jersey heifers (10 - 12 months of age) in Louisiana was initially evaluated in 1985 and monitored over a 1-year period, which covered breeding age and gestation (Boddie et al., 1987). Samplings from the teat skin, teat canal, and mammary secretion were performed bimonthly and continued through time of calving. For bacteriologic analysis, a total of 388 samples from teat skin, 388 from teat canal keratin, and 216 from secretions were examined; not all quarters contained sufficient volumes of secretions.

For teat skin, staphylococci were isolated on mannitol salt agar, and the predominant colony type of a particular isolate based on color, rough/smooth, size, hemolytic pattern, and appearance was collected, sub-plated for culture on blood agar, and identified to the species level (Boddie et al., 1987). *Staphylococcus xylosus* (20.9%) and *Staph. chromogenes* (14.9%) were the predominant flora isolated from teat skin, followed by *Staph. warneri* (6.7%), *Staph. sciuri*  (6.2%), *Staph. aureus* (2.8%), *Staph. hyicus* (1.3%), and *Staph. simulans* (1.3%). The most prevalent bacteria found in teat canals were *Staph. chromogenes* (41.0%), followed by *Staph. hyicus* (16.8%), *Staph. aureus* (10.0%), *Staph. xylosus* (1.0%), *Staph. warneri* (0.8%), and *Staph. sciuri* (0.5%).

In mammary secretions, *Staph. chromogenes* (49.5%) was the predominant organism, followed by *Staph. hyicus* (21.3%), *Staph. aureus* (13.0%), *Streptococcus uberis* (1.4), and *Staph. xylosus* (0.9%). The CNS species in teat canals and in secretions from a particular mammary quarter were present at the first sampling, and the same CNS species were isolated from teat canals and secretions from the same individual quarters with each successive sampling of the trial. Also, there appeared to be a correlation between colonization of the teat canal and IMI. For example, the major CNS species colonizing the teat canal (*Staph. chromogenes*  (41.0%), *Staph. hyicus* (16.8%), *Staph. aureus* (10.0%), and *Staph. xylosus* (1.0%)) were also the predominant organisms causing IMI (*Staph. chromogenes* (49.5%), *Staph. hyicus* (21.3%), *Staph. aureus* (13.0%), and *Staph. xylosus* (0.9%)). Thus, teat canals infected with *Staph. chromogenes* (41.0%) were positively correlated with secretions (49.5%) infected with these bacteria. The overall correlation between teat canal colonization and IMI was 82.2% across these 4 CNS species.

This initial study (Boddie et al., 1987) indicated that teat skin, teat canals, and mammary secretions of heifers are colonized with CNS as well as *Staph. aureus* at an early age, and that infections may persist for up to 1 year. Species identification demonstrated that nearly all isolates from the same quarter throughout the study were the same biovariant based on the API Staph-Ident System as previously modified (Watts et al., 1984), which supports the contention that isolates from each quarter over time were from persistent infections and not from new IMI occurring between sampling periods. Although the percentage of *Staph. aureus* isolates was lower than CNS, presence of this major pathogen demonstrated that it colonized teat canals at a much earlier age than documented previously (Rendel & Sundberg, 1962).

334 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

(20%). Mixed isolates of CNS and *Streptococcus* species were also found.

secretions.

*sciuri* (0.5%).

species.

freshening with clinical mastitis. Subsequent study of breeding age animals in a research herd revealed that IMI may be diagnosed as early as 6 months of age, and infections persisted throughout pregnancy and into lactation (Boddie et al., 1987). Other studies demonstrated that greater than 90% of breeding age and bred heifers (12 - 24 months of age) may be infected (Trinidad et al., 1990b). Most of the infections were shown to be caused by the CNS (*Staphylococcus chromogenes* and *Staphylococcus hyicus*) followed by *Staph. aureus*

The prevalence of mastitis in 10 unbred Jersey heifers (10 - 12 months of age) in Louisiana was initially evaluated in 1985 and monitored over a 1-year period, which covered breeding age and gestation (Boddie et al., 1987). Samplings from the teat skin, teat canal, and mammary secretion were performed bimonthly and continued through time of calving. For bacteriologic analysis, a total of 388 samples from teat skin, 388 from teat canal keratin, and 216 from secretions were examined; not all quarters contained sufficient volumes of

For teat skin, staphylococci were isolated on mannitol salt agar, and the predominant colony type of a particular isolate based on color, rough/smooth, size, hemolytic pattern, and appearance was collected, sub-plated for culture on blood agar, and identified to the species level (Boddie et al., 1987). *Staphylococcus xylosus* (20.9%) and *Staph. chromogenes* (14.9%) were the predominant flora isolated from teat skin, followed by *Staph. warneri* (6.7%), *Staph. sciuri*  (6.2%), *Staph. aureus* (2.8%), *Staph. hyicus* (1.3%), and *Staph. simulans* (1.3%). The most prevalent bacteria found in teat canals were *Staph. chromogenes* (41.0%), followed by *Staph. hyicus* (16.8%), *Staph. aureus* (10.0%), *Staph. xylosus* (1.0%), *Staph. warneri* (0.8%), and *Staph.* 

In mammary secretions, *Staph. chromogenes* (49.5%) was the predominant organism, followed by *Staph. hyicus* (21.3%), *Staph. aureus* (13.0%), *Streptococcus uberis* (1.4), and *Staph. xylosus* (0.9%). The CNS species in teat canals and in secretions from a particular mammary quarter were present at the first sampling, and the same CNS species were isolated from teat canals and secretions from the same individual quarters with each successive sampling of the trial. Also, there appeared to be a correlation between colonization of the teat canal and IMI. For example, the major CNS species colonizing the teat canal (*Staph. chromogenes*  (41.0%), *Staph. hyicus* (16.8%), *Staph. aureus* (10.0%), and *Staph. xylosus* (1.0%)) were also the predominant organisms causing IMI (*Staph. chromogenes* (49.5%), *Staph. hyicus* (21.3%), *Staph. aureus* (13.0%), and *Staph. xylosus* (0.9%)). Thus, teat canals infected with *Staph. chromogenes* (41.0%) were positively correlated with secretions (49.5%) infected with these bacteria. The overall correlation between teat canal colonization and IMI was 82.2% across these 4 CNS

This initial study (Boddie et al., 1987) indicated that teat skin, teat canals, and mammary secretions of heifers are colonized with CNS as well as *Staph. aureus* at an early age, and that infections may persist for up to 1 year. Species identification demonstrated that nearly all isolates from the same quarter throughout the study were the same biovariant based on the API Staph-Ident System as previously modified (Watts et al., 1984), which supports the In a subsequent herd survey (Trinidad et al., 1990b), the prevalence of mastitis in breeding age and pregnant heifers was determined in 4 commercial dairies. Teat canal keratin and secretion samples were collected from 116 Jersey heifers, and results revealed that teat canal colonizations were present in 93.1% of heifers and 70.7% of quarters. *Staph. aureus* was isolated from teat canal keratin samples of 31% of heifers and 12.3% of quarters. Other organisms isolated from keratin and percentage frequencies were *Staph. chromogenes* (42.9%), *Staph. hyicus* (25.2%), other staphylococcal species (5.7%), *Strep. dysgalactiae* (0.6%), S*trep.*  species (3.1%), and mixed isolates containing staphylococci and streptococci (5.7%). In this 4 herd survey, IMI were found in 96.9% of heifers and in 74.6% of quarters. Twenty-nine percent of heifers and 15.1% of quarters showed clinical symptoms of mastitis as evidenced by clots, flakes, and blood. *Staph. aureus* was isolated from 14.7% of quarters. This microorganism was also isolated from 25% of quarters with clinical symptoms. *Staph. aureus* causes severe damage to mammary tissue (Trinidad et al., 1990a), and infections are very difficult to eliminate in lactating cows. Other organisms isolated from secretions and percentage frequencies were *Staph. chromogenes* (43.1%). *Staph. hyicus* (24.3%), other staphylococcal spp. (3.6%), *Strep. dysgalactiae* (0.4%), *Strep.* spp. (3.3%), *Nocardia* species (0.4%), and mixed isolates containing staphylococci and streptococci (5.1%).

These initial studies on heifer mastitis were performed in Louisiana, USA, where a warm and humid climate with a long fly season may be conducive to IMI in these young dairy animals. Thus, a subsequent national trial was carried out using 1583 breeding age heifers from 28 herds to determine the prevalence of heifer mastitis in the states of California, Washington, and Vermont as well as in Louisiana (Fox et al., 1995). The majority of quarter infections were caused by the CNS (mean = 27.1%). The mean prevalence of *Staph. aureus* among the 4 sites was 2.9%, but was highest in Louisiana at 10.1% during the spring season. The overall IMI prevalence was greatest in Louisiana, and the highest frequency was during the 3rd trimester of gestation just prior to parturition, which was due to an increase in IMI cause d by *Staph. aureus*, CNS, and environmental streptococci. Likewise, following parturition, the greatest prevalence of IMI was in Louisiana, which had the greatest percentage of heifers with IMI caused by the environmental streptococci and *Staph. aureus*, and the 2nd greatest prevalence of CNS among the 4 sites. For breeding age and bred heifers, the stage of pregnancy significantly affected IMI prevalence, which was highest for heifers in the last trimester of pregnancy, especially CNS IMI. This study by Fox et al. (1995) showed that site location had a significant effect on prevalence of mastitis, and that Louisiana had the greatest prevalence of IMI. This was postulated to be due to the warm and humid climate, as well as to a prolonged horn fly season in Louisiana that exposed udders and teats to an increased bacterial load that management practices failed to control.

## **3. Mammary leukocyte response to intramammary infection**

In lactating cows, the milk somatic cell count (SCC) composed of leukocytes (macrophages, lymphocytes, neutrophils) and a small percentage of mammary epithelial cells, is considered an important parameter for assessing mammary health status (e.g. inflammation); milk yield decreases as SCC and incidence of mastitis increase. Thus, SCC in breeding age and pregnant heifer mammary gland secretions have been analyzed to measure the degree of inflammation and potential reductions in future milk yield. In a study by Boddie et al. (1987), the mean arithmetic SCC of quarters from unbred heifers infected with *Staph. chromogenes, Staph. hyicus*, and *Staph. aureus* were 7.8, 8.5, and 9.2 x 106/ml, respectively, whereas the mean SCC of uninfected quarters was 3.5 x 106. The mean SCC of heifer secretions collected on the day of freshening were 3.2 x 106/ml and 1.6 x 106/ml for quarters infected by staphylococci and uninfected quarters, respectively. The mean SCC during the first 3 months of lactation in quarters infected with *Staph. chromogenes, Staph. hyicus*, and *Staph. aureus* were 168, 193, and 578 x 103/ml, respectively, while the SCC of uninfected quarters was 39 x 103/ml. Thus, this study found SCC approaching 200 x 103 for quarters infected with CNS during the first 3 months of lactation, and, based on previous studies (Jones et al., 1984; Kirk, 1984), SCC in this range are associated with milk loss. Approximately 13% of quarter secretions sampled prepartum contained *Staph. aureus*, and after freshening, the SCC of these quarters averaged 578 x 103/ml, a cell count that has been associated with a loss of >4.4 lb (2.0 kg) of milk/day (Kirk, 1984).

Other studies have also demonstrated elevated SCC in heifer mammary glands infected with mastitis-causing bacteria. For example, in a study by Trinidad et al. (1990b), average arithmetic SCC in secretions from 325 quarter samples regardless of infection status was 11.7 x 106/ml. Infected (n = 240) and uninfected (n = 85) quarters had secretion SCC of 13.6 x 106/ml and 5.7 x 106/ml, respectively. Of the staphylococci, *Staph. aureus*-infected quarters had the highest secretion SCC/ml (17.3 x 106), followed by *Staph. chromogenes* (12.8 x 106), and *Staph. hyicus* (12.4 x 106). The mean SCC for non-agalactiae streptococci was 15.5 x 106/ml.

The volume of mammary secretion is very low in breeding-age animals; thus, SCC become concentrated, resulting in high SCC even in uninfected quarters. However, SCC approach 20 x 106/ml in quarters infected with *Staph. aureus,* and over 13.6 x 106/ml in those infected with the CNS and *Strep.* species. Such elevated SCC over a long period of time suggests that mammary tissue in affected quarters are in a state of chronic inflammation, which could adversely affect development of milk-producing tissues and negatively affect future milk yield. In response to infection, neutrophils become the major leukocyte type that infiltrates mammary tissue from the vascular system. The function of the neutrophil influx via chemotaxis is to phagocytose and kill mastitis-causing bacteria. But there is also evidence that this defense response can impair and disrupt mammary function (Akers & Thompson 1987; Capuco et al., 1986; Zhao & Lacasse, 2008). For example, in addition to the damage caused by bacterial toxins, the migration of leukocytes, namely neutrophils, across the mammary epithelial surfaces is thought to cause mechanical damage and/or chemical damage (via release of reactive oxygen species) to the milk secretory cells as well as to the ductal cells of the mammary gland.

## **4. Histological response of the mammary gland to IMI**

336 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

milk/day (Kirk, 1984).

106/ml.

**3. Mammary leukocyte response to intramammary infection**

In lactating cows, the milk somatic cell count (SCC) composed of leukocytes (macrophages, lymphocytes, neutrophils) and a small percentage of mammary epithelial cells, is considered an important parameter for assessing mammary health status (e.g. inflammation); milk yield decreases as SCC and incidence of mastitis increase. Thus, SCC in breeding age and pregnant heifer mammary gland secretions have been analyzed to measure the degree of inflammation and potential reductions in future milk yield. In a study by Boddie et al. (1987), the mean arithmetic SCC of quarters from unbred heifers infected with *Staph. chromogenes, Staph. hyicus*, and *Staph. aureus* were 7.8, 8.5, and 9.2 x 106/ml, respectively, whereas the mean SCC of uninfected quarters was 3.5 x 106. The mean SCC of heifer secretions collected on the day of freshening were 3.2 x 106/ml and 1.6 x 106/ml for quarters infected by staphylococci and uninfected quarters, respectively. The mean SCC during the first 3 months of lactation in quarters infected with *Staph. chromogenes, Staph. hyicus*, and *Staph. aureus* were 168, 193, and 578 x 103/ml, respectively, while the SCC of uninfected quarters was 39 x 103/ml. Thus, this study found SCC approaching 200 x 103 for quarters infected with CNS during the first 3 months of lactation, and, based on previous studies (Jones et al., 1984; Kirk, 1984), SCC in this range are associated with milk loss. Approximately 13% of quarter secretions sampled prepartum contained *Staph. aureus*, and after freshening, the SCC of these quarters averaged 578 x 103/ml, a cell count that has been associated with a loss of >4.4 lb (2.0 kg) of

Other studies have also demonstrated elevated SCC in heifer mammary glands infected with mastitis-causing bacteria. For example, in a study by Trinidad et al. (1990b), average arithmetic SCC in secretions from 325 quarter samples regardless of infection status was 11.7 x 106/ml. Infected (n = 240) and uninfected (n = 85) quarters had secretion SCC of 13.6 x 106/ml and 5.7 x 106/ml, respectively. Of the staphylococci, *Staph. aureus*-infected quarters had the highest secretion SCC/ml (17.3 x 106), followed by *Staph. chromogenes* (12.8 x 106), and *Staph. hyicus* (12.4 x 106). The mean SCC for non-agalactiae streptococci was 15.5 x

The volume of mammary secretion is very low in breeding-age animals; thus, SCC become concentrated, resulting in high SCC even in uninfected quarters. However, SCC approach 20 x 106/ml in quarters infected with *Staph. aureus,* and over 13.6 x 106/ml in those infected with the CNS and *Strep.* species. Such elevated SCC over a long period of time suggests that mammary tissue in affected quarters are in a state of chronic inflammation, which could adversely affect development of milk-producing tissues and negatively affect future milk yield. In response to infection, neutrophils become the major leukocyte type that infiltrates mammary tissue from the vascular system. The function of the neutrophil influx via chemotaxis is to phagocytose and kill mastitis-causing bacteria. But there is also evidence that this defense response can impair and disrupt mammary function (Akers & Thompson 1987; Capuco et al., 1986; Zhao & Lacasse, 2008). For example, in addition to the damage caused by bacterial toxins, the migration of leukocytes, namely neutrophils, across the mammary epithelial surfaces is thought to cause mechanical damage and/or chemical Initially, the mammary glands of dairy heifers were studied to determine histological responses to teat canal colonization with *Staph. chromogenes* and *Staph. hyicus* (Boddie et al. (1987). Two unbred heifers were slaughtered, one at 8 months and the other at 18 months of age, and examination of mammary tissues from both heifers demonstrated a leukocyte reaction to the colonization of the teat canal. Cross-sections through the mid-teat canal demonstrated cocci colonizing keratinized cells of the canal lumen, and sections of distal teat cisternal tissues demonstrated heavy leukocyte infiltration with lymphocytes and plasma cells at Fürstenberg's rosette compared with uninfected tissues.

Subsequently, 7 unbred heifers, 14 - 26 months of age, were studied to evaluate the effects of IMI on leukocyte infiltration and characteristics of secretory tissue in developing mammary glands (Trinidad et al., 1990a). Histologic observations of tissue samples from lobes of mammary parenchyma of uninfected quarters showed that alveoli were small; the epithelial lining was composed of a single layer of cuboidal cells surrounding a small luminal space with little or no stained secretory product (See Figures 1 through 3). Interalveolar connective tissue area composed approximately half of the observed lobes, and a few infiltrating leukocytes, mainly lymphocytes, were observed.

Infected tissues, particularly those with *Staph. aureus* IMI, exhibited large amounts of interalveolar connective tissues and reductions in epithelial and luminal areas (See Figures 4-8). Such areas also exhibited leukocytic infiltration, particularly lymphocytes and neutrophils, into stromal and luminal areas. Hyperplasia of ducts and cisterns as a result of infection was also observed, and macro- and microscopic abscesses were found in the parenchyma of one quarter infected with *Staph. aureus*. Abscesses were tubercule-like with a circular, stratified fibrosis containing numerous lymphocytes, neutrophils, plasma cells, and multinucleated giant cells.

Results of morphometric analysis on parenchymal tissue components showed that percentages of each component in uninfected quarters were very similar to percentages from quarters infected with CNS, although quarters infected with CNS exhibited significantly more stromal area. Percentages of alveolar epithelium and lumen in quarters infected with *Staph. aureus* were significantly lower (P < .05) than those in uninfected quarters and in quarters infected with CNS. Quarters infected with *Staph. aureus* also showed a greater percentage (P < .05) of interalveolar stroma than did uninfected quarters and quarters infected with CNS. Thus, *Staph. aureus*-infected tissue demonstrated reduced secretory activity. The greatest development of secretory tissue in young heifers occurs during the first pregnancy, and developing secretory tissues may be affected adversely by bacterial infection and inflammation, leading to deposition of connective tissue stroma instead of milk secretory tissue and a subsequent deleterious effect on future milk yield.

**Figure 1.** General view of a cross-section of a lobe of mammary tissue from an uninfected quarter exhibiting large ducts (D) and undeveloped lobules of parenchyma (P) among adipose tissue stroma. (A). x18.

**Figure 2.** Portion of mammary parenchymal tissue typical of that obtained from uninfected quarters and those infected with coagulase-negative staphylococci exhibiting small alveoli with empty, ovoid lumens (1) and those with some secretions (2). x180.

Managing Mastitis in Heifers: An Initial Step in Improving Dairy Herd Health 339

338 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**Figure 1.** General view of a cross-section of a lobe of mammary tissue from an uninfected quarter exhibiting large ducts (D) and undeveloped lobules of parenchyma (P) among adipose tissue stroma.

**Figure 2.** Portion of mammary parenchymal tissue typical of that obtained from uninfected quarters and those infected with coagulase-negative staphylococci exhibiting small alveoli with empty, ovoid

lumens (1) and those with some secretions (2). x180.

(A). x18.

**Figure 3.** Portion of uninfected parenchymal tissue revealing limited stroma (S), flattened epithelium (E), and distended luminal areas (L) engorged with flocculent matter suggested active secretion. x180.

**Figure 4.** Parenchymal tissue from a quarter infected with *Staph. aureus* exhibiting a large interalveolar connective tissue stroma (S) and limited alveolar luminal areas (L). D = Duct. x180. Trinidad et al., 1990a.

**Figure 5.** Parenchymal tissue from a quarter infected with *Staphylococcus aureus* showing numerous neutrophils (arrows) infiltrating a luminal area (L) of one alveolus. x500.

**Figure 6.** Extensive epithelial hyperplasia (H) was observed in ductal linings in the parenchyma from one quarter infected with *Staphylococcus aureus. Lymphoid cells* (arrowheads) were numerous in the epithelium as well as in the underlining connective tissue. D = duct. x180.

Managing Mastitis in Heifers: An Initial Step in Improving Dairy Herd Health 341

340 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**Figure 5.** Parenchymal tissue from a quarter infected with *Staphylococcus aureus* showing numerous

**Figure 6.** Extensive epithelial hyperplasia (H) was observed in ductal linings in the parenchyma from one quarter infected with *Staphylococcus aureus. Lymphoid cells* (arrowheads) were numerous in the

epithelium as well as in the underlining connective tissue. D = duct. x180.

neutrophils (arrows) infiltrating a luminal area (L) of one alveolus. x500.

**Figure 7.** Abscess (A) from one quarter infected with *Staphylococcus aureus* exhibiting tubercle-like morphology with circular stratified fibrosis (arrows) and marked cellular infiltration. E = Portion of enlarged in Figure 8. P = Parenchyma. x18.

**Figure 8.** Magnification of an edge of the abscess shown in Figure 7. Neutrophils (arrows), macrophages (arrowheads), and multinucleated giant cells (MGC) were present in this area of the abscess. x500. Trinidad et al., 1990a.

Leukocyte infiltration into cisternal and parenchymal mammary tissues was also evaluated. Quarters infected with *Staph. aureus* exhibited the greatest tissue leukocytosis, followed by quarters infected with CNS and uninfected quarters. Leukocyte infiltration in gland cistern and secretory tissue for infected quarters was significantly higher (P < .05) than that for uninfected quarters. Leukocytosis into teat cistern tissue was similar for uninfected quarters and those infected with CNS, but significantly lower (P < .05) than quarters with *Staph. aureus* IMI. None of the uninfected quarters or quarters infected with CNS demonstrated marked leukocyte infiltration. However, marked leukocyte infiltration, particularly lymphocytes, into cisternal and parenchymal areas was commonly observed in quarters that were infected with *Staph. aureus*. The majority of leukocytes observed within alveolar lumina were neutrophils.
