Chronic mastitis (CM) is generally considered for cows having high somatic cell count (SCC) >200 000 cells/ml for more than two consecutive months or by the occurrence of three or more repeated cases of clinical mastitis in the same mammary quarter during lactation (Cardozo et al., Reference Cardozo, Thaler Neto, Souza, Picinin, Felipus, Reche, Schmidt, Werncke and Simon2015). Cows with CM have low chance of clinical cure (Cardozo et al., Reference Cardozo, Thaler Neto, Souza, Picinin, Felipus, Reche, Schmidt, Werncke and Simon2015) and a high frequency of mammary tissue fibrosis, which results in permanent milk yield losses. Considering the growing concern regarding use of antimicrobials and animal welfare issues, previous studies evaluated less aggressive, non-antimicrobial alternatives for drying-off quarters during lactation, such as casein hydrolyzate (CH) (Shamay et al., Reference Shamay, Shapiro, Mabjeesh and Silanikove2002). Intramammary infusion of CH mimics the natural mammary involution that occurs at the end of lactation, through the production of peptides associated with decreased milk yield and involution of the mammary gland after drying-off (Shamay et al., Reference Shamay, Shapiro, Leitner and Silanikove2003; tho Seeth et al., Reference tho Seeth, Wente, Paduch, Klocke, Hoedemaker and Krömker2016).
Although previous studies evaluated the efficacy of intramammary infusion of CH at quarters drying-off with CM, the effects of the frequency of CH infusions have not yet been evaluated. Previous studies have used multiple infusions (Shamay et al., Reference Shamay, Shapiro, Mabjeesh and Silanikove2002; Shamay et al., Reference Shamay, Shapiro, Leitner and Silanikove2003; Silanikove et al., Reference Silanikove, Iscovich, Leitner and Hogeveen2005; tho Seeth et al., Reference tho Seeth, Wente, Paduch, Klocke, Hoedemaker and Krömker2016), which increases the risk of injury to the teat canal and new intramammary infections, and additionally these repeated procedures are more time consuming. Thus, the aim of the present study was to evaluate the efficacy of two CH intramammary infusion protocols during lactation on composite SCC and milk yield. The hypothesis was that a single intramammary infusion of 300 mg of CH would have a similar effect as three intramammary infusions of 100 mg of CH at drying-off mammary quarters with CM during lactation.
Materials and methods
Ethics approval was obtained from the Ethical Committee on the Use of Animals of the School of Veterinary Medicine and Animal Science, University of São Paulo (CEUA, FMVZ, USP – Brazil/SP, protocol number 1876290119).
Inclusion criteria and cases definition
Sixty Holstein and Girolando (crossbred Bos taurus taurus × Bos taurus indicus) cows (approximately 250 d in milk (DIM) and milk yield of approximately 20 kg/d) that had only one quarter with CM (clinical and subclinical) and four functional mammary quarters were selected. Definition of clinical and subclinical CM, as well as individual cow information about parity, DIM, average milk yield before treatment, clinical mastitis history, and last composite SCC are available in online Supplementary Table S1.
Intramammary casein hydrolyzate infusion
The preparation of CH was performed according to Shamay et al. (Reference Shamay, Shapiro, Leitner and Silanikove2003). Selected cows were randomly distributed between two treatment groups: (a) three intramammary CH infusions (three-CH; 100 mg, 50 ml per infusion), with 24-h intervals or (b) single intramammary infusion of CH (one-CH; 300 mg, 50 ml). In both treatment groups, an internal teat sealant infusion (Sellat®, Ourofino Saúde Animal, Cravinhos, Brazil) was infused at day 7 after the last CH infusion. Prior to intramammary CH infusion, teats were immersed in a pre-milking disinfectant solution and dried after 30 s with disposable paper towels, followed by complete milking. Teat ends asepsis was performed with 70% iodized alcohol solution (70% alcohol + 2% iodine), followed by infusions of CH. At the end of the CH infusion, the quarter was immersed in a post-milking disinfectant solution.
Milk samples and analysis
Milk samples from selected quarters were collected before (day 0) and after (days 4 and 7) the CH infusions for identification of mastitis-causing pathogen and for SCC. The microbiological identification was performed by MALDI-TOF MS according to Barcelos et al. (Reference Barcelos, Martins, Grenfell, Juliano, Anderson, dos Santos and Gonçalves2019). The SCC was determined by flow cytometry using an electronic optical counting equipment (Somacount 300®, Bentley Instruments Inc., Chaska, MN USA). Milk yield was measured by the automatic milk yield meters in each farm before the beginning of treatment (day 0) and at day 7 in order to evaluate the difference in milk production after CH infusions. The cessation of lactation was assessed using the udder pressure index (UPI) methodology, based on manual assessment of udder pressure on days 4 and 7 after the first CH infusion (Leitner et al., Reference Leitner, Jacoby, Maltz and Silanikove2007).
Milk samples from the untreated quarters (pool of the three functional quarters) were collected at 30, 60 and 90 d after treatments to determine the composite SCC of CH treated cows. The results were compared with SCC of the four quarters obtained before to treatment.
Detailed description of sample collection procedure, MALDI-TOF MS analysis and UPI methodology are available in the online Supplementary File.
Statistical analysis
Data were analyzed using the statistical software SAS version 9.4 (SAS Institute, Cary, NC, USA). Statistical significance was defined when P-value ≤ 0.05. The adjusted means of the descriptive characteristics of the selected cows before infusion of CH, as well as the results of milk SCC were compared between treatments by Tukey test. For cessation of lactation index the PROC GLIMMIX was used. Somatic cell count ( × 103 cells/ml) was transformed to logarithmic scale according to Schukken et al. (Reference Schukken, Wilson, Welcome, Garrison-Tikofsky and Gonzalez2003). Analysis of milk yield data (D0 and D7) and SCC of untreated quarters (D30, D60 and D90) was performed by PROC MIXED. The model included treatment, day, their interaction, SCC log prior to treatment, CM history, DIM and parity as fixed effects and farm as random effect.
Results and discussion
Cow characteristics
Of the cows included (n = 60), 38 (63.33%) were Holstein and 22 (36.67%) Girolando. Parity, milk yield, SCC (composite and per quarter), clinical mastitis history and DIM before the first CH infusion were similar (P > 0.05) between treatment groups (details in Supplementary Table S1). Selected cows had a higher parity and advanced DIM, which are risk factors described in cases of CM. Of the 60 milk samples collected for SCC analysis prior to treatments, 11 were not evaluated because they were coagulated, and not acceptable for the analysis of flow cytometry. Thus, 25 quarters treated with three-CH and 24 quarters treated with one-CH were evaluated for SCC and were included in the study.
Frequency of mastitis causing pathogens
Of the total quarters evaluated (n = 60), 17 (28.33%) had negative culture before treatments. Negative culture results may occur because the number of colonies forming units is below the limit of detection of microbiological culture, there has been spontaneous bacterial cure or the causative agent is not isolated in conventional culture media. Among the positive samples, Streptococcus uberis had the highest frequency of isolation followed by Staphylococcus aureus. The ability of these pathogens to cause CM may be associated with bacterial defense mechanisms that contribute to immune system evasion.
After CH infusions (D7), of the total quarters evaluated (n = 60), 10 quarters (16.67%) presented bacteriological cure (three-CH = 6 mammary quarters; one-CH = 4). Although the cure rate after seven days of CH application was low, it is expected that postpartum cure rate will be higher (Silanikove et al., Reference Silanikove, Iscovich, Leitner and Hogeveen2005).
Evaluation of cessation of lactation using udder pressure
Mammary quarters treated with three-CH had a higher degree of udder pressure compared to those treated with one-CH (P < 0.001). In addition, a significant effect of CH treatment protocol on the day of evaluation was observed (P < 0.01, Fig. 1 and online Supplementary Fig. S2). Nevertheless, no complete quarter cessation of lactation (score 0) was observed in either treatment within 7 d of evaluation. This was not expected, since in previous studies with intramammary infusion of CH, both in chronic cases of lactation and for cows at drying-off, a complete cessation of lactation of quarter was reported in most cows (Silanikove et al., Reference Silanikove, Iscovich, Leitner and Hogeveen2005; tho Seeth et al., Reference tho Seeth, Wente, Paduch, Klocke, Hoedemaker and Krömker2016). Although our study did not observe a complete cessation of lactation of the mammary gland (UPI score 0), no quarter showed swelling (score 3) after treatment, which was considered a positive effect of CH infusion. Similar results were also described by Leitner et al. (Reference Leitner, Jacoby, Maltz and Silanikove2007), which found that CH reduced udder pressure and was associated with more comfort compared to cows that used only dry cow therapy.
Fig. 1. Frequency of udder pressure scores of the mammary quarters treated with one or three intramammary casein hydrolyzate infusions for days 4 and 7 after treatment. Udder pressure scores range from 0 (no pressure, lactation has ceased) to 3 (significant pressure, full lactation). For a full statistical evaluation refer to online Supplementary Fig. S2.
Milk yield
The milk yield was measured only in five farms, totalizing 45 cows evaluated (one-CH = 21 and three-CH = 24). Milk yield data from 15 cows were not available for analyses due to the lack of farm production records. Milk yield of cows treated with one-CH decreased from 21.65 to 18.77 kg, while that of cows treated with three-CH decreased from 21.14 to 17.09 kg. The average milk yield was only affected by DIM and evaluation day (both P > 0.001). On average, the difference in milk yield before (D0) and after treatment (D7) was 2.9 kg (13.45% of pre-infusion milk yield). The decrease of total milk yield was greater than that reported in other studies that evaluated the loss of cow's milk yield when a quarter was dried off with CH infusions. While in the Silanikove et al. (Reference Silanikove, Iscovich, Leitner and Hogeveen2005) study, the total cow's milk yield was not affected after drying-off a quarter, Leitner et al. (Reference Leitner, Koren, Jacoby, Merin and Silanikove2012) reported a reduction of approximately 9% in milk yield 30 d after mammary quarter drying-off. However, the milk yield reduction was lower than that reported by Skarbye et al. (Reference Skarbye, Krogh and Sørensen2018) who, when evaluating the abrupt quarter drying-off with subclinical mastitis, observed an average reduction of 4.1 kg of milk. This result may suggest improved comfort conditions for CH-infused cows, since there was no increase in udder pressure after the interruption of the quarter milking.
SCC of untreated mammary quarters
Of all cows selected (n = 60), 28 remained in lactation for at least 90 d after CH infusions and were evaluated for SCC of composite milk samples of untreated quarters on days 30, 60 and 90. Thirty-two cows were not included, in which 16 were dried-off by proximity of calving, 10 were dried-off for other reasons (reproductive problems and low milk yield) and six were voluntary culled by herd owners from the herd. There was no difference of SCC of composite milk samples between CH treatment protocols at any of the evaluated days (P > 0.05). For cows treated with three-CH, SCC was significantly lower only at day 60 (P < 0.05), which is similar to the results described by Silanikove et al. (Reference Silanikove, Iscovich, Leitner and Hogeveen2005), while cows treated with one-CH had significantly lower SCC on days 30, 60 and 90 (P < 0.01 or better) when compared to the results of SCC evaluated before CH infusion (Fig. 2 and online Supplementary Fig. S3). Our results should be interpreted carefully because we did not have a control group. Moreover, future studies should evaluate the effect of CH in chronic clinical cases and chronic subclinical cases separately, also testing single doses below 300 mg of CH and postpartum effects in the subsequent lactation.
Fig. 2. Effect of casein hydrolyzate infusion in a single mammary quarter with chronic mastitis on log SCC in milk sampled from the other healthy quarters. For a full statistical evaluation refer to online Supplementary Fig. S3.
In conclusion, the protocol with one-CH has the potential to be used as an alternative method for drying-off mammary quarters with CM during lactation, but further studies are needed considering that intramammary therapy with CH constitutes an unapproved use in commercial dairy farms.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029921000467
Acknowledgements
We are grateful to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; grant number 001) for the scholarship support.
