Blitz therapy in control of Streptococcus agalactiae subclinical mastitis in dairy cows reduces somatic cells count of bulk milk tank in a dairy cattle herd

Article history The objective of this study was to assess the control feasibility of Streptococcus agalactiae subclinical mastitis and its impact in reducing somatic cell counts (SCC) of bulk milk tank. During the lactation, 33 cows positive for Streptococcus agalactiae in microbiological culture were treated with three applications of intramammary antimicrobial containing 100.000 UI of penicillin and 150 mg of novobiocin, in an interval of 12 hours. Individual and bulk tank milk samples were collected for somatic cell count (SCC) analysis and milk yield was measured before and after treatment. Statistical analyses were performed with SAS System, version 9.2. All cows treated against S. agalactiae had microbiological cure and reduction of SCC, indicating the effectiveness of treatment. SCC in bulk tank was reduced from 829,000 to 513,000 cells/ml. Received 15 June 2016 Received in revised form 15 December 2016 Accepted 07 February 2017


INTRODUCTION
Mastitis caused by Streptococcus agalactiae is extremely costly once these bacteria usually infect many quarters of the mammary gland, causing extensive damage to alveolar cells and resulting in decrease of milk production (EDMONDSON, 2011;KEEFE, 2012).The reduction of intramammary infection rates, and consequently the reduction in somatic cell count (SCC), adds great economic value to the milk produced, and allows great profit to the farmers due to the improvement of dairy industries efficiency, also ensuring quality and safety of dairy products offered to consumers (KLEI; YUN, 1998).
The reduction of total bacterial count (TBC) and SCC has become a common goal since the implementation of programs to improve milk quality and the need of dairy industries of high milk quality (BRASIL, 2012;ALVES et al., 2014).In this context, the subclinical mastitis treatment during the lactation can be considered an alternative in herds with high prevalence of S.agalactiae infection (TYLER et al., 1992;REYES et al., 2015).In these herds, selecting cows eligible to the blitz therapy (i.e. the antibiotic intramammary treatment into all four quarters during lactation to eradicate S. agalactiae infections) through microbiological culture proved to be extremely important and economically feasible (EDMONDSON, 2011;REYES et al., 2015).Additionally, individual SCC and microbiological cultures are essential to monitor prevalence of infections and to evaluate treatments efficacy.
This study aimed to evaluate technical feasibility of controlling S. agalactiae and its impact on the quality of milk as SCC, using the treatment of infected cows during the lactation, i.e., blitz therapy.

MATERIALS AND METHODS
The Animal Ethics Committee of the Universidade Federal de Minas Gerais (protocol n. 117/2011) approved the protocols for this study.
The study was conducted on a dairy farm in the state of Minas Gerais, in a herd with 114 crossbred Holstein x Gyr lactating cows with an average production of 18 kg milk/day.The general management of milking included good hygiene practices and proper review of the milking equipment.
Individual milk samples from all lactating cows were collected for microbiological culture in days 21, 14 and 7, and the day of the treatment was considered day zero.Milk samples were obtained immediately before milking and put into a sterile container, after proper antiseptic procedures at the teat end.Collected material was frozen and sent to laboratory and microbiological culture was done according to Brito and Brito (1999).
Volumes of 10 µl of each sample were seeded with a calibrated, disposable handle in each quadrant of a blood agar plate with 5% of sheep's blood without fibrin, following 35ºC incubation during 24 hours, according to Harmon et al. (1990).After the incubation period, reading of first plates was carried out, noting the microbial growth, aspect, color and number of occurring colonies.Subsequently, plates were again incubated at 35ºC for another 24 hours, performing the second reading after a total incubation period of 48 hours.Milk samples presenting growing microorganisms, working as a representative colony, were selected, seeded in agar BHI (Brain Heart Infusion -brain and heart agar infusion) and incubated at 35ºC for 24 hours.After this period, the isolated microorganisms were microscopically examined with smears colored by Grams technique, and evaluated regarding its catalase production and, then, submitted to identification tests.

Identification of positive coco gram and negative catalase
This group was identified via the CAMP test (Christie, Atkins e Munch-Peterson), sodium hipurato hydrolysis, growth in presence of 6.5 of sodium chloride, growth in environment containing bile, aesculin and aesculin hydrolysis.The classification was carried out as per Brito and Brito (1999).
Among the 40 animals identified as carriers of S. agalactiae, 33 received three consecutive intramammary treatments, with an interval of 12 hours, 10 ml of associated 100.000UI of penicillin and 150 mg of novobiocin, with prior teats antisepsis performed with cotton soaked in alcohol 70%.The milk produced by these animals was discarded according to the withdrawal period.
Drying off was carried out early in the other seven animals identified as being in an advanced stage of lactation.All treated animals were segregated after treatment, including milking period, until the last result of microbiological culture.As the number of animals identified in the microbiological exam with S. aureus was too small (n = 2), they were culled.
Microbiological cultures of milk from treated animals were performed at days 14 and 21.Animals were considered cured if no growth of S. agalactiae was observed in culture results after treatment.Individual SCC analysis from all lactating cows was performed at days 7, 14, and 21.Bulk tank milk samples were collected for SCC analysis on days 30 and 21, according to Brito (2001).

Statistical analysis
'Blitz therapy' effect over time on the individual's somatic cell counts was assessed using a mixed model approach, in order to adequately take into account the dependence among observations over time.Counts were first logtransformed in order to stabilize the variance and obtain approximate normality.
The model employed was Y_jt = μ+τ_t+e_jt, where Y_jt is the ln(SCC) for the jth cow at the tth time period; μ is the overall mean; τ_t is the effect of the tth day relative to treatment; and e_jt is the random error associated with the jth cow at tth time point measurement, assumed to be normally distributed with zero mean, constant variance and covariance given by a first-order autoregressive (AR(1)) structure.This was selected among other common covariance structures (variance components, compound symmetry, unstructured, banded main diagonal, first-order autoregressive, heterogeneous AR(1), first-order ante-dependence, heterogeneous CS and spatial power) by both AIC and BIC criteria, either considering or not the cow effect as random, following the general guidelines given by Milliken and Johnson (2009).
Once the best fitted covariance structure was selected, the effect of time over ln(SCC) was assessed by means of Type 3 F test and ln(SCC) by means of least squares method for 14 and 21 days after treatment application.They were compared with ln(SCC) LS' mean 7 days before the application through the Dunnett-Hsu test, a test designed for comparisons with a control (LITTELL et al., 2006).
The analyses were performed with the MIXED procedure of SAS System, version 9.2 (SAS Institute Inc., 2009) and the graphs were created in R software, version 3.0.0(R Core Team, 2013).Statistical significance was considered when p ≤ 0.05.

RESULTS
Thirty-three animals infected with S. agalactiae received intramammary antimicrobial treatment and their milk was discarded following recommended withdrawal period.Microbiological culture results from 114 animals enrolled in the study are shown in Table 1.All animals treated with the blitz therapy (n = 33) showed no S. agalactiae growth at microbiological culture performed on days 14 and 21 after treatment.'Blitz therapy' was highly effective in reducing SCC when comparing average somatic cell count at day 7 before the application with the count means of days 14 and 21 after that application (p < 0.0001 for both comparisons), as it can be clearly seen on Figure 1.SCC in bulk tank assessed the impact of blitz therapy in the months before and after the treatment.SCC in bulk tank, which was 829,000 cells/ml in the month before the blitz therapy, i.e. during a high prevalence of S. agalactiae period, was reduced to 513,000 cells/ml after 30 days of treatment.Milk production three months before and three months after the blitz therapy did not change significantly (p > 0.05).

DISCUSSION
All animals treated with blitz therapy (n = 33) showed no growth of S. agalactiae in microbiological culture performed at days 14 and 21.Control of S. agalactiae can be difficult because the infection spreads quickly among cows if the milking hygiene, milking routines or milking machine performance is less than optimal.However, this study presents encouraging results, which can be seen as an alternative tool in order to eradicate S. agalactiae in herds with a high prevalence of this pathogen, because the therapy allows animals to be treated during the lactation.As a main advantage, blitz therapy has a fast reduction of subclinical infections and SCC, compared to practices of pre and post dip and dry cow therapy, which results, although excellent, can only be seen in a long term (ERSKINE; EBERHART, 1990).Smith and Ward (1975) reported an infection reduction of 21% to 4.7% in quarters positive for S. agalactiae, 20 days after treatment using intramammary penicillin.Goodger et al. (1988) obtained microbiological cure in 97 out of 99 cows treated with cephapirin after three weeks.Efficacy of antibiotics treatment depends on three factors: proper concentration of drug on the site of infection, susceptibility of microorganism to the drug, and host´s intact defenses to fight infection (VIANA, 2000).Results show that three consecutive intramammary applications using penicillin and novobiocin were enough to keep antimicrobial minimum inhibitory concentration within the mammary gland, insuring drug's action against the organism and increasing the chances of microbiological cure.
Blitz therapy presents overall very good results for eradicating S. agalactiae in positive herds; however, this practice should not be seen as the only solution to prevent mastitis but as an auxiliary method to integrated programs of mastitis control (GREEN, 2000).Teat disinfection after milking and dry cow therapy was implemented simultaneously to treatment during lactation.It is also important to adopt hygienic practices such as pre and post dip, drying teats with disposable paper towels, adequate milking equipment maintenance and cleaning, and dry cow therapy, if not earlier, at least simultaneously to implementation of blitz therapy.
In this study, treatment of cows carrying S. agalactiae resulted in significant reduction of SCC in bulk tank.Similar results were seen in a study conducted by Goldberg et al. (1991) in a herd with SCC average of 918,000 cells/ml before blitz therapy; it was reduced to 439,000 cells/ml 30 days after treatment.Before treatment, 40 animals positive for S. agalactiae were responsible for 47.04% of SCC in bulk tank milk; this value was reduced to 18.48% after removing S. agalactiae, proving the role of this agent in the high incidence of SCC in the herd.
Even though the impact of S. agalactiae in milk production is well known, a high variability of the effects of blitz therapy on milk production can be seen.Studies on the effect of blitz therapy in milk production indicate great benefits of this treatment to animals in early lactation.Yagamata et al. (1987) obtained 98% of cure rate for S. agalactiae and demonstrated a better cost-benefit for treated cows in early and mid-lactation, with earnings of US$ 396 and US$ 237, respectively.For treated cows in late lactation, the benefit was comparatively lower, US$ 55.However, these authors did not consider the impact of these infected animals by contagious agents in the new intramammary infections in the herd, since they are the main source of infection in healthy animals (TYLER et al., 1992;EDMONDSON, 2011).Reduction of clinical mastitis and non-intentional culling of animals, increase of milk production in next lactation and reduction of SCC in bulk tank should be taken into consideration while deciding whether to perform the therapy, mainly in programs with payment for quality.
Mastitis control programs using milking hygiene practices and good herd management to reduce the prevalence of S. agalactiae may be more economically feasible than the blitz therapy, but in cases when these programs are not effective in reducing the prevalence of S. agalactiae, treatment during lactation is reasonable (EDMONDSON, 2011).Given the reduction in percentage of SCC in bulk milk tank of treated cows, it is possible to evaluate the impact of S. agalactiae in milk quality.Blitz therapy should be considered in cases in which industries' penalties regarding high SCC are economically representative, when the prevalence of infected animals and losses related to milk production are high, as well as in institutional programs, either public or private, of SCC control in specific regions.
Farms undertaking eradication of S. agalactiae need to monitor the progress of the control, especially in 'clean' cows groups, which have been segregated or treated, in order to get early warning of re-emerging problems.New infection rates, provided by individual somatic cell counts, are key in monitoring the efficacy of those treatments.

CONCLUSION
Use of Blitz Therapy to control subclinical mastitis caused by S. agalactiae proved to be technically feasible, resulting in microbiological cure of cows and reduction of SCC at days 14 and 21 post treatment.Use of Blitz Therapy to control subclinical mastitis caused by S. agalactiae reduced the somatic cell count of bulk tank.

Figure 1 -
Figure 1 -Somatic cell count (SCC) profiles for the 33 cows considered in the study: (a) counts, (b) log of counts.

Table 1 -
Microbiological culture results from 114 animals enrolled in the study.