**10. Role of vaccination in mastitis control**

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

normally during the later stages of pregnancy.

cured of IMI.

**treatment**

to be effective for quarters infected with CNS but waiting until this time to treat chronic *Staph. aureus* mastitis might be too late. A mammary gland that has been infected with *Staph. aureus* for several months to a year will not develop normally, and treatment during the immediate prepartum period would most likely be of little benefit in curing infections or salvaging mammary tissue. At this point, the tissue damage would have already been done, and affected quarters should have been treated earlier in gestation to: 1) cure existing infections; 2) reduce chronic inflammation; and 3) allow mammary tissue to develop

Results of these trials demonstrated that nonlactating and lactating cow antimicrobial treatment of heifers known to be at risk for developing IMI is advantageous because the cure rate is much higher than that obtained when treating infections during lactation. In addition, most studies showed that SCC are lower, there is no milk loss due to therapy , risk of antibiotic residue at calving is minimal, and future milk production is increased in heifers

A disadvantage of prepartum lactating cow antibiotic therapy for controlling mastitis in heifers is the potential for antibiotic residues, especially if heifers calve sooner than expected. In one study (Oliver et al., 1992), it was shown that 17% of colostrum samples from heifer mammary glands infused with cloxacillin were positive for antibiotic residues by the *Bacillus stearothermophilus* disc assay, the majority of which were from heifers that calved within 5 days of treatment. Only 4.5% of samples obtained at the first milking after parturition were positive for antibiotic residues if intramammary infusion of cloxacillin occurred > 7 days before parturition. All samples obtained 3 days after parturition, the time when milk would likely be marketed for human consumption, were negative for antibiotic residues. In contrast, 85% of colostrum samples and 28.2% of samples obtained 3 days after parturition were positive for cephapirin residues, and marked variability between time of antibiotic treatment and parturition with persistence of antibiotic residues was observed. Thus, antibiotic treatment of heifer mammary glands earlier in gestation may be advantageous from a residue standpoint, but the timing of antibiotic treatment and

Another study was conducted to determine if antibiotic treatment of heifer mammary glands earlier in the prepartum period reduced occurrence of residues in milk (Oliver et al., 1997a). A total of 82 Jersey heifers was used. Approximately half served as negative controls (n = 42) and half received an intramammary infusion of 200 mg cephapirin sodium (n = 40) 14 days before calving. Forty percent of samples from cephapirin-treated quarters were positive at the first milking after calving, but only 3.1% of samples obtained from antibiotictreated quarters at the sixth milking (3 days) after calving were positive; 3 of the 4 positive

**9. Antibiotic residues in milk following prepartum lactating cow** 

subsequent persistence in mammary secretions could impact efficacy.

samples were from a heifer that calved early and within 3 days of treatment.

Although antimicrobial therapy is successful, the goal from a herd management perspective is to prevent new infections from occurring, and vaccination has been attempted as a prophylactic measure. Recent research has demonstrated that several experimental *Staph. aureus* vaccines, as well as one commercial vaccine, can increase antistaphylococcal antibody titers and reduce the new infection rate in heifers. A *Staph. aureus* vaccine formulated to stimulate pseudocapsule and alpha toxin antibodies was evaluated in heifers in New York (Sears et al., 1990). At 4 and 2 weeks prior to calving, heifers were given subcutaneous injections into the supramammary lymph node, and after calving, heifers were challenged with *Staph. aureus*. Vaccinates demonstrated a 52% reduction in new IMI. In addition, 64% of intramammary infections in control cows became chronic compared with only 12% in vaccinates.

A field study in Norway evaluated a *Staph. aureus* vaccine that contained two strains of whole, formalin-inactivated bacteria with pseudocapsule, alpha and beta toxoids, and mineral oil as an adjuvant (Nordhaug et al., 1994). A total of 108 pregnant heifers on 16 farms with an average *Staph. aureus* prevalence of 19.2% was used. Vaccinates were injected subcutaneously in the area of the supramammary lymph node with a dose of 2.5 ml at 8 and 2 weeks before calving. Results showed a 46% reduction in new IMI during the subsequent lactation. Antibody titers to *Staph. aureus* pseudocapsule and alpha toxin were markedly elevated in the serum of vaccinates, and these titers remained significantly higher in serum and milk during the entire lactation compared with those of unvaccinated controls.

In Argentina, a vaccine was developed based on an inactivated, encapsulated *Staph. aureus*  strain, a crude extract of *Staph. aureus* exopolysaccharides, and inactivated, unencapsulated *Staph. aureus* and *Streptococcus* species in an aluminum hydroxide adjuvant (Giraudo et al., 1997). This formulation was evaluated in three groups of ten 24- to 26-month-old heifers each in a 7-month trial. The first group received an intramuscular injection of the vaccine in the neck at 8 and 4 weeks prepartum, the second group was vaccinated similarly at 1 and 5 weeks postpartum, and a third group (control) received placebo injections at 8 and 4 weeks prepartum. The research herd from which the heifers were selected had a bulk tank SCC ranging from 480,000 to 730,000, and 19% of quarters were infected with *Staphylococcus aureus*. This immunization program showed that the frequency of new *Staph. aureus*  infection was reduced from 18.8% in controls to 6.7 and 6.0% for heifers vaccinated

prepartum and postpartum, respectively; the protective effect was maintained for at least 6 months.

In view of more recent studies showing success of vaccines in heifers, researchers in Louisiana evaluated a commercially available *Staph. aureus* vaccine in young dairy animals (Nickerson et al., 1999). The vaccine was a lysed culture of polyvalent *Staph. aureus* somatic antigens representing 5 phage types in an aluminum hydroxide adjuvant base, including serotypes 5, 8, and 336, the most common *Staph. aureus* serotypes associated with clinical mastitis (Lysigin®, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA). At 6 months of age, 35 Jersey heifers were vaccinated using a 5-ml dose intramuscularly in the semimembranosus muscle of the rear leg, and 14 days later, vaccinates received a booster dose, which was repeated at 6-month intervals. Another 35 heifers served as unvaccinated controls. Results demonstrated that: (1) the number of quarters exhibiting chronic intramammary infection during pregnancy was reduced 43.1% in vaccinates compared with controls; (2) rate of new intramammary infection during pregnancy was reduced 44.8%; (3) rate of new intramammary infection at freshening was reduced 44.7%; and (4) the SCC was reduced by 50% in vaccinates compared with controls.

In a subsequent, more in depth study using the same vaccine (Lysigin®), 106 Holstein heifers from the James River Correctional Center dairy herd Goochland, VA, USA were evaluated (Nickerson et al., 2009). This herd had a 9,979-kg rolling herd average milk production with an average SCC of ~200,000/ml. Previous microbiological culture of heifer mammary secretions indicated that approximately 35% of animals were infected with *Staph. aureus*.

At approximately 6 to 18 months of age, heifers were processed through a restraining chute to collect aseptic quarter mammary secretion samples for microbiological. Fifty-three heifers were vaccinated using a dose of 5 ml intramuscularly that was administered as above, and the other 53 heifers served as unvaccinated controls. Fourteen days after the initial processing, the vaccinated group was again processed through the chute and boosted with Lysigin®. All animals were maintained on pasture and rotated by age group through calving. At 6-month intervals after the initiation of the trial and through time of calving, the vaccinated group was again processed through the chute for boosting.

At 2-month intervals after the trial initiation and through calving, mammary secretion samples were collected for bacteriological culture and for the determination of electronic SCC (A/SN Foss, Hillerod, Denmark). Microbiological examination of quarter samples collected from bred heifers over gestation demonstrated that 19.8% of heifers (9.4% of quarters) were infected with *Staph. aureus*, 68.9% of heifers (34.3% of quarters) were infected with CNS, 6.6% of heifers (2.3% of quarters) were infected with environmental streptococci, and 1% of heifers (0.3% of quarters) were infected with coliforms.

At time of calving, heifers were enrolled in the Dairy Herd Improvement Program (DHIA) and data were recorded for milk yield, percentages and actual pounds fat and protein, days in milk, and SCC. Data on vaccine efficacy were examined in terms of mean percentage reduction in rate of new *Staph. aureus* or CNS intramammary infections achieved among immunized heifers compared with the rate among unimmunized controls at the time of calving; differences between the percentage of heifers becoming infected among treatments was tested with the standard normal approximation.

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

reduced by 50% in vaccinates compared with controls.

months.

prepartum and postpartum, respectively; the protective effect was maintained for at least 6

In view of more recent studies showing success of vaccines in heifers, researchers in Louisiana evaluated a commercially available *Staph. aureus* vaccine in young dairy animals (Nickerson et al., 1999). The vaccine was a lysed culture of polyvalent *Staph. aureus* somatic antigens representing 5 phage types in an aluminum hydroxide adjuvant base, including serotypes 5, 8, and 336, the most common *Staph. aureus* serotypes associated with clinical mastitis (Lysigin®, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA). At 6 months of age, 35 Jersey heifers were vaccinated using a 5-ml dose intramuscularly in the semimembranosus muscle of the rear leg, and 14 days later, vaccinates received a booster dose, which was repeated at 6-month intervals. Another 35 heifers served as unvaccinated controls. Results demonstrated that: (1) the number of quarters exhibiting chronic intramammary infection during pregnancy was reduced 43.1% in vaccinates compared with controls; (2) rate of new intramammary infection during pregnancy was reduced 44.8%; (3) rate of new intramammary infection at freshening was reduced 44.7%; and (4) the SCC was

In a subsequent, more in depth study using the same vaccine (Lysigin®), 106 Holstein heifers from the James River Correctional Center dairy herd Goochland, VA, USA were evaluated (Nickerson et al., 2009). This herd had a 9,979-kg rolling herd average milk production with an average SCC of ~200,000/ml. Previous microbiological culture of heifer mammary secretions indicated that approximately 35% of animals were infected with *Staph. aureus*.

At approximately 6 to 18 months of age, heifers were processed through a restraining chute to collect aseptic quarter mammary secretion samples for microbiological. Fifty-three heifers were vaccinated using a dose of 5 ml intramuscularly that was administered as above, and the other 53 heifers served as unvaccinated controls. Fourteen days after the initial processing, the vaccinated group was again processed through the chute and boosted with Lysigin®. All animals were maintained on pasture and rotated by age group through calving. At 6-month intervals after the initiation of the trial and through time of calving, the

At 2-month intervals after the trial initiation and through calving, mammary secretion samples were collected for bacteriological culture and for the determination of electronic SCC (A/SN Foss, Hillerod, Denmark). Microbiological examination of quarter samples collected from bred heifers over gestation demonstrated that 19.8% of heifers (9.4% of quarters) were infected with *Staph. aureus*, 68.9% of heifers (34.3% of quarters) were infected with CNS, 6.6% of heifers (2.3% of quarters) were infected with environmental streptococci,

At time of calving, heifers were enrolled in the Dairy Herd Improvement Program (DHIA) and data were recorded for milk yield, percentages and actual pounds fat and protein, days in milk, and SCC. Data on vaccine efficacy were examined in terms of mean percentage reduction in rate of new *Staph. aureus* or CNS intramammary infections achieved among immunized heifers compared with the rate among unimmunized controls at the time of

vaccinated group was again processed through the chute for boosting.

and 1% of heifers (0.3% of quarters) were infected with coliforms.

Immunization with Lysigin® did not cause any adverse reactions at the injection site or systemically. Minimal swelling (<2.5 cm) was occasionally observed, which disappeared within 48 hours of administration. Vaccine efficacy data showed that the percentage of heifers with *Staph. aureus* intramammary infections at freshening was lower in vaccinates (13.3%) compared with controls (34.0%); a reduction of 60.9% (P < 0.01). Likewise, an examination of health records showed that the percentage of heifers that were culled or died during the trial was reduced by approximately one-third by vaccination: 16.9% in vaccinates and 24.5% in controls (P > 0.05). The vaccinated group also experienced a slight, insignificant reduction in mastitis caused by CNS. At freshening, intramammary infections with CNS were lower in vaccinates (64.2%) compared with controls (69.8%); a reduction of 8.1%.

Somatic cell counts in samples collected during first week of lactation irrespective of infection status were 45% lower in vaccinates compared with controls (287,317 vs. 522,345/ml). Somatic cell counts in samples collected during first week of lactation from uninfected heifers for vaccinates and controls were 66,095 and 132,754/ml, respectively; a 50.2% reduction; and for infected heifers, SCC were 441,764 and 892,176/ml, respectively; a 50.5% reduction. Somatic cell counts in samples collected during the prepartum period were highest for *Staph. aureus* (6,730 x 103), followed by the environmental streptococci (3,850 x 103), and CNS (3,510 x 103).

An examination of the 305-day lactation milk yield for the 1st lactation of both vaccinated and unvaccinated control heifers demonstrated an approximate 8.6% increase in vaccinates vs. controls (11,217 vs. 10,332 kg, respectively) or a difference of 886 kg. On a complete lactation basis, vaccinated animals produced 839 kg more milk than controls (12,537 vs. 11,698 kg, respectively); an increase of 7.3%.

The percentage of 305-day lactation fat was higher in vaccinates than controls (3.64 vs. 3.27%, respectively); however, the percentage of 305-day lactation protein was slightly higher in controls than vaccinates (3.06 vs. 2.95, respectively). Actual 305-day kilograms of both fat and protein were higher in vaccinates than controls (fat: 408 vs. 339 kg, respectively; protein: 330 vs. 315 kg, respectively). Likewise, on a complete lactation basis, actual kilograms of both fat and protein were higher in vaccinates than controls (fat: 460 vs. 393, respectively; protein: 370 vs. 353, respectively).

An examination of the number of days in milk for the first lactation demonstrated that vaccinates persisted 13 days longer than the unvaccinated controls (349 vs. 336 days). In addition, average first lactation SCC were 11,000 cells/ml lower in vaccinates compared with controls (49,000 vs. 60,000/ml).

Results of this Virginia investigation demonstrated that vaccinating dairy heifers according to the prescribed protocol with a commercial USDA licensed *Staph. aureus* bacterin, Lysigin®, reduced the number of new *Staph. aureus* intramammary infections at time of calving by 60.9%, lowered the SCC by 50%, and decreased the culling rate by approximately one-third.

In addition, overall milk yield, production of fat and protein, and number of days in milk were greater in vaccinated heifers compared with controls.

The decrease in frequency of new *Staph. aureus* intramammary infections at calving (60.9%) in vaccinates using Holstein heifers is higher than the 44.7% reduction observed in a Louisiana trial using the same vaccine in Jersey heifers (Nickerson et al., 1999). In both trials, SCC at calving were reduced by approximately 50%. The 60.9% efficacy found in the present trial is also higher than the 40.2% efficacy observed by Giraudo et al. (1989), the 46% efficacy observed by Nordhaug et al. (1994), and the 52% efficacy observed by Sears et al. (1990). However, it is difficult to compare among the latter three trials as the vaccine formulations were all quite different.

The question becomes: Is it economically feasible to use this commercial vaccination protocol on young dairy heifers? Based on an average of 1,864 more lb milk per vaccinated heifer, which translates to 18.64 hundredweights (cwt) of milk (1,864/100), at the current (2008) price of \$25.00/cwt, an increased income of \$466.00/heifer would be realized (18.64 cwt x \$25.00/cwt = \$466.00). If each heifer was vaccinated beginning at 6 months of age until calving, this would entail vaccinations at 1) 6 months, 2) a booster 2-weeks later, and subsequently at 3) 12 months, 4)18 months, and 5) 24 months, or a total of 5 immunizations through calving. At \$1.50/dose, this cost would total \$7.50, which when subtracted from the increased income from milk production, would yield a net income of \$458.50 per heifer (\$466.00 - \$7.50). This figure does not include the costs of labor involved in the immunization process; however, it is evident that vaccination is well worth the cost of the vaccine. Not only does it reduce new infections in first calf heifers at parturition, it may also reduce the introduction of *Staph. aureus* to the milking herd.

It is obvious that use of experimental and commercially available *Staph. aureus* vaccines can be used to prevent new infections, especially when used in heifers. Efficacy has been shown to range between 44 to 61% with reductions in SCC of 50%. This prevention strategy may represent a major control mechanism for managing *Staph. aureus* in the future, especially as new antigens and adjuvants are added to vaccine preparations.
