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Effects of bovine casein hydrolysate as a dry cow therapy on prevention and cure of bovine intramammary infection, milk production, and somatic cell count in the subsequent lactation

  • Author Footnotes
    * These authors contributed equally to this work.
    Ezra Shoshani
    Correspondence
    Corresponding author
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Mileutis Ltd., 7 Golda Meir St., Nes Ziona 7403650, Israel
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  • Author Footnotes
    * These authors contributed equally to this work.
    M. van Straten
    Footnotes
    * These authors contributed equally to this work.
    Affiliations
    Hachaklait–Mutual Society for Cattle Insurance and Veterinary Services in Israel Ltd., 20 Bareket St., Industrial Park, Caesaria 388900, Israel
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  • Author Footnotes
    * These authors contributed equally to this work.
Open AccessPublished:March 10, 2022DOI:https://doi.org/10.3168/jds.2021-20703

      ABSTRACT

      The primary objectives were to investigate the efficacy of bovine casein hydrolysate (bCNH) as a dry cow therapy at (1) preventing new intramammary infection (IMI) postpartum of all bacteria and coagulase-negative staphylococci (CNS), and (2) curing existing subclinical infections, mainly of CNS. The secondary objective was to measure the effects of bCNH on milk yield, composition, and somatic cell count (SCC) during the lactation postcalving. The trial was conducted as a randomized, blinded controlled experiment. Israeli Holstein dairy cows (n = 170) in first or higher lactations were recruited from 4 large commercial dairy herds. Cows were enrolled following clinical examination and bacteriological sampling of each quarter, which was the experimental unit. Random allocation was implemented at the cow level. All quarters of 100 cows were treated with 1 dose of bCNH (60 mg diluted in 20 mL of sterile solution) and those of 70 control cows were treated with saline solution. Clinical assessment of each cow's general appearance, teat-end leakage, and teat morphology was performed for 0, 1, 2, 3, 7, and 14 d after treatment, together with follow-up clinical observation and clinical examination of udder quarters. Quarter aseptic milk samples were obtained for bacteriological culture 48 h pretreatment, at time of treatment, and 3 and 5 d postcalving. Multivariable analyses were conducted to study the effects of bCNH on cure and prevention of IMI, adjusting for parity, farm, average of daily milk yield for 305 d, and average of monthly SCC values for 305 d of previous lactation. The odds of preventing IMI in cows treated with bCNH at dry-off were 2.15 times higher [95% confidence interval (CI): 1.15 to 4.00] than in cows treated with saline. Prevention was mostly of CNS. The odds of preventing CNS in cows treated with bCNH at dry-off were 2.20 times higher (95% CI: 1.58 to 3.07) than in control cows. The odds of curing IMI caused by CNS in cows treated with bCNH at dry-off were 4.80 times higher (95% CI: 0.75 to 30.75) than in saline-treated cows. Log SCC, adjusted to that of the previous lactation, was lower in the bCNH group compared with controls for 305 d in milk postcalving. The average milk yield per day for 305 d, adjusted to average daily milk yield of previous lactation, was higher by 2.1 kg in the bCNH group compared with controls (95% CI: 1.21 to 3.20). Clinical assessment of udders and cows posttreatment showed no negative effects of bCNH. Incidence of stillbirth, clinical mastitis, retained placenta, endometritis (5 to 12 d postcalving), ketosis, abortions, and reproduction did not differ between the 2 groups. Results suggest that a single intramammary administration of bCNH at dry-off effectively increases milk yield and lowers SCC, prevents new IMI during the dry period, and may be a beneficial alternative for curing existing IMI at dry-off, mainly by CNS.

      Key words

      INTRODUCTION

      In dairy cows, a dry period is essential for good udder health and desirable productivity in the subsequent lactation (
      • Capuco A.V.
      • Akers R.M.
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      • Vanhoeij R.J.
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      • Kemp B.
      • Lam T.J.G.M.
      The effect of dry period length and antibiotic treatment at drying off on somatic cell counts across the dry period.
      ;
      • Rajala-Schultz P.J.
      • Gott P.N.
      • Proudfoot K.L.
      • Schuenemann G.M.
      Effect of milk cessation method at dry-off on behavioral activity of dairy cows.
      ). In many countries, drying off is performed by abrupt cessation of milking in conjunction with dry cow therapy (DCT), which is based on applying antimicrobial substances on all quarters to treat subclinical infection and prevent new IMI (
      • Natzke R.P.
      • Everett R.W.
      • Bray D.R.
      Effect of drying off practices on mastitis infection.
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      Control of milk secretion and apoptosis during mammary involution.
      ;
      • National Mastitis Council
      Dry Cow Therapy.
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      • Dohoo I.R.
      • Roy J.P.
      • Keefe G.P.
      Evaluation of selective dry cow treatment following on-farm culture: Risk of postcalving intramammary infection and clinical mastitis in the subsequent lactation.
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      • Lacasse P.
      • Ordonez-Iturriaga A.
      • Francoz D.
      Non-antimicrobial approaches at drying-off for treating and preventing intramammary infections in dairy cows. Part 1. Meta analyses of efficacy of using an internal teat sealant without a concomitant antimicrobial treatment.
      ). However, DCT is not effective against all pathogens (
      • Oliver S.P.
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      Impact of antibiotic use in adult dairy cows on antimicrobial resistance of veterinary and human pathogens: A comprehensive review.
      ;
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      • Verbeke J.
      • De Vliegher S.
      Infection dynamics across the dry period using dairy herd improvement somatic cell count data and its effect on cow performance in the subsequent lactation.
      ). Selective DCT for cows with IMI was first suggested by
      • Philpot W.N.
      Role of therapy in mastitis control.
      and later evaluated by others (
      • Woolford M.W.
      • Williamson J.H.
      • Day A.M.
      • Copeman P.J.A.
      The prophylactic effect of a teat sealer on bovine mastitis during the dry period and the following lactation.
      ;
      • Cameron M.
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      • Roy J.P.
      • Keefe G.P.
      Evaluation of selective dry cow treatment following on-farm culture: Risk of postcalving intramammary infection and clinical mastitis in the subsequent lactation.
      ;
      • Scherpenzeel C.G.M.
      • den Uijl I.E.M.
      • van Schaik G.
      • Olde Riekerink R.G.M.
      • Keurentjes J.M.
      • Lam T.J.G.M.
      Evaluation of the use of cow antibiotics in low somatic cell count cows.
      ;
      • Swinkels J.M.
      • Hilkens A.
      • Zoche-Golob V.
      • Krömker V.
      • Buddiger M.
      • Jansen J.
      • Lam T.J.G.M.
      Social influences on the duration of antibiotic treatment of clinical mastitis in dairy cows.
      ), with or without internal teat sealant.
      Following dry-off, milk accumulation leads to an increase in intramammary pressure, which can cause milk leakage from the teat canal and impair keratin formation (
      • Dingwell R.T.
      • Leslie K.E.
      • Schukken Y.H.
      • Sargeant J.M.
      • Timms L.L.
      • Duffield T.F.
      • Keefe G.P.
      • Kelton D.F.
      • Lissemore K.D.
      • Conklin J.
      Association of cow and quarter-level factors at drying-off with new intramammary infections during the dry period.
      ;
      • Sordillo L.M.
      Mammary gland immunology and resistance to mastitis.
      ), thereby increasing the risk of IMI. During this time, the risk of microorganism entry into the gland through the teat canal is high (
      • Leelahapongsathon K.
      • Piroon T.
      • Chaisri W.
      • Suriyasathaporn W.
      Factors in dry period associated with intramammary infection and subsequent clinical mastitis in early postpartum cows.
      ) and is more pronounced in high-producing dairy cows (
      • Rajala-Schultz P.J.
      • Hogan J.S.
      • Smith K.L.
      Association between milk yield at dry-off and probability of intramammary infections at calving.
      ;
      • Zobel G.
      • Leslie K.
      • Weary D.
      • Von Keyserlingk M.
      Gradual cessation of milking reduces milk leakage and motivation to be milked in dairy cows at dry-off.
      ). Involution of the bovine mammary gland is complete after around 4 wk (
      • Capuco A.V.
      • Akers R.M.
      Mammary involution in dairy animals.
      ;
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ). At this time, liquid secretion is rich in SCC, lactoferrin, IgG, H2O2, and NO (
      • Bushe T.
      • Oliver S.P.
      Natural protective factors in bovine mammary secretions following different methods of milk cessation.
      ;
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ), teat-end integrity improves, and teat-canal closure is enhanced. Thus, physiological processes combined with higher bactericidal and bacteriostatic characteristics of the gland dramatically decrease the risk of acquiring new infections beyond this period.
      A novel approach aimed at hastening mammary gland involution and enhancing teat-canal closure through an immunological mechanism during the dry period would be of benefit in decreasing new IMI in the dry period and increasing the comfort and health of the dairy cow. The immunomodulator bovine casein hydrolysate (bCNH) can rapidly induce the innate immune system by mimicking the gland's natural involution process (
      • Shamay A.
      • Shapiro F.
      • Mabjeesh S.J.
      • Silanikove N.
      Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.
      ;
      • Silanikove N.
      • Iscovich J.
      • Leitner G.
      Therapeutic treatment with casein hydrolyzate eradicates effectively bacterial infection in treated mammary quarters in cows.
      ;
      • Ponchon B.
      • Lacasse P.
      • Silanikove N.
      • Ollier S.
      • Zhao X.
      Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution.
      ;
      • Zhao X.
      • Ponchon B.
      • Lanctot S.
      • Lacasse P.
      Invited review: Accelerating mammary gland involution after drying-off in dairy cattle.
      ). Tight junctions are erupted and influx of macrophages, neutrophils, lymphocytes, BSA, lactoferrin, matrix metalloproteinase 2, and matrix metalloproteinase 9 is initiated, followed by their rapid increase in concentration. Intramammary administration of 6 doses of bCNH over 3 consecutive days accelerates mammary gland involution to between 3 to 5 d after treatment (
      • Shamay A.
      • Shapiro F.
      • Mabjeesh S.J.
      • Silanikove N.
      Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.
      ,
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ;
      • Silanikove N.
      • Iscovich J.
      • Leitner G.
      Therapeutic treatment with casein hydrolyzate eradicates effectively bacterial infection in treated mammary quarters in cows.
      ), concurrent with the aforementioned changes in the tight junctions. Consequently, bCNH shows broad-spectrum antimicrobial activity against contagious pathogens, such as Staphylococcus aureus, as well as environmental pathogens, such as Escherichia coli (
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ).
      Due to the efficacy of bCNH after multidose administration in controlling IMI, we investigated whether, under field conditions, a single intramammary dose of 60 mg of bCNH diluted in 20 mL of sterile solution, administered at dry-off would (1) prevent the establishment of new IMI during the dry period and in the first 10 d of the subsequent lactation, (2) cure infections from the previous lactation, and (3) increase milk yield in the subsequent lactation concurrent with a reduction in SCC.

      MATERIALS AND METHODS

      Trial Approval

      A negatively controlled, blinded randomized clinical trial was conducted using dairy cattle from commercial dairy farms in Israel. The clinical study was carried out following a grant for clinical research of bCNH during drying off by the Animal Experimentation Ethics committee of the Agricultural Research Organization–Volcani Center, Ministry of Agriculture and Rural Development, and Pharmaceutical Division of the Ministry of Health, Israel.

      Herd Selection and Characteristics

      Four farms were included in the study with approximately 3,167 lactating dairy cows. The farms were in different geographical areas, at different altitudes, and with different annual rainfall amounts (Table 1). In the 4 geographical areas, maximum average temperature ranged from 24°C to 30°C (April–August), and minimum average temperature ranged from 11°C to 20°C (April–August) for the study period (source: Israel Meteorological Service). More descriptive details of the farms participated in this trial are presented in Table 1.
      Table 1Studied dairy farm characteristics and management features
      ParameterNa'amanTzvaimGivat Haim MeuhadDarom
      Shed
       Shed typeLoose housingLoose housingFree stallsLoose housing
       Area/milking cow (m2)20201016
       Area/dry cow (m2)15251723
       Separate shed for high SCC cowsNoNoNoYes
      Milking
       TypeHerringboneCarouselCarouselPolygon
       Milking stations (n)20282828
       Milkings/d3333
       LocationNorthwestEastCenterSouth
       Average rainfall (mm)700500500300
       Milking cows (n)1,140754540733
       Average milk (kg, 305 d)11,20910,87010,22711,422
       Dry cow treatmentBlanketBlanketBlanketBlanket
       Dry-off procedureAbruptAbruptAbruptAbrupt
      Cooling was provided in the summer months by constant ventilation in the sheds. During the hot season (May–September), milking cows and cows in preparation for calving (2 wk before calving) were cooled in a separate shed with ventilators and water sprinklers in cycles of 5 min each, which included 30 s of water spraying at a water capacity of 300 L/h, and 4.5 min of close ventilation. Milking cows were cooled 6 times a day, and preparation cows 3 times a day on most farms. Dry cows were not cooled with sprinklers.
      The bedding area of the milking cow sheds, except for those in Givat Haim Meuhad (with freestalls), was cultivated daily to a depth of 40 cm. Dairy farms generally have separate sheds for milking cows and dry cows, the latter divided into dry cows and cows in preparation for calving (2 wk before calving). On one farm, there was a separate group for high SCC cows or cows with contagious pathogens. These cows were the last group to be milked in the milking facility. Female calves were separated from their mothers no later than 2 h postcalving and were raised in a separate area of the dairy farm.
      Farms were selected according to the following criteria: participation in the Herd Health Program of “Hachaklait” (Mutual Society for Cattle Insurance and Veterinary Services), likelihood of complying with the study protocol, absence of bovine leukemia virus, member of the Israeli Cattle Breeders Association (ICBA), average bulk milk SCC <250,000 cells/mL during the previous 12 mo (Table 1), sufficiently large total herd size to dry-off 15 to 20 cows/wk, participation in a monthly testing schedule (such as that of the DHIA; i.e., measurements of individual cow SCC, milk butterfat, protein and lactose contents, and milk weight), and having consistent and accurate recordings of clinical mastitis, production, reproduction, culling, and death events. These data, along with the management, health, production, and reproduction data, were recorded to the farm computer using NOA (a herd-management program in Hebrew; ICBA), and national dairy herd-management software (ICBA). All clinical examinations during the study were performed by Hachaklait veterinarians.
      Actual drying off was performed once or twice weekly, depending on season and herd size. Cows were milked 3 times a day in a milking facility that varied among farms (Table 1). Milk liners were either round or triangular. After each milking, the milking systems were cleaned and washed with dilute active chloride and inorganic bases. The milking systems were also cleaned every 3 to 7 d with phosphorous acid.

      Study Cow Selection, Randomization, and Allocation to Treatment Groups and Procedures to Ensure Blinding

      Cows participating in the study were of the Israeli Holstein–Friesian breed. The average number of lactations per cow was 3.2. Eligible cows were first randomly enrolled to the trial according to their programmed dry-off date (i.e., between April 2009 and July 2009) and later according to the inclusion and exclusion criteria. The enrolled process ended when the accumulated number of cows reached the target of 195. Cows were destined for dry-off in the study if they appeared healthy, as determined by clinical examination, had 4 functional quarters, and were free of teat lesions or abnormalities. In addition, they were only included in the study if they had not received systemic or intramammary antimicrobial or anti-inflammatory therapy in the 4 wk before dry-off, were pregnant, and were not intended for culling after calving.
      Random allocation was implemented at the cow level, using a restricted randomization method in which treatments were randomly assigned within blocks of 17 cows, keeping a 10:7 ratio for bCNH treatment and negative control, respectively. The block of 17 cows was determined by selecting cows from all herds according to this ratio. Recruitment to either control group or bCNH group was performed using the PLAN procedure (SAS version 9.2, SAS Institute Inc.). A computer-generated randomization list was created by the monitor. This list was filed in a separate folder marked “for the treatment technician's eyes only” and given to the treatment technicians on each farm. These technicians allocated the next available number coding the destined treatment to each recruited cow upon entry into the trial and treated the cow accordingly. The treatment technicians were the only ones who were allowed to view the allocation tables. The code was not revealed to the technicians who evaluated the cows and udders before and after treatment, or who sampled the milk, or any other personnel participating in the trial. Similarly, treatment technicians were not allowed to participate in or be present at any of the evaluations or milk samplings.
      As shown in Figure 1, data from 161 cows (644 quarters) were available for analysis. For the analysis of prevention of an IMI, a quarter had to test negative from any bacteria on both pretreatment bacteriological tests; 461 quarters (72% of all eligible quarters) met this criterion. In addition, 4 quarters with contaminated samples in posttreatment bacteriological tests were removed from the analysis. Thus, the final database for this analysis included 457 quarters (192 for the control group and 265 for the bCNH group).
      Figure thumbnail gr1
      Figure 1Cow flow diagram [adapted from Consolidated Standards of Reporting Trials (CONSORT);
      • Moher D.
      • Schulz K.F.
      • Altman D.G.
      The CONSORT statement: Revised recommendations for improving the quality of reports of parallel-group randomized trials.
      ]. bCNH = bovine casein hydrolysate.
      Each participating cow was identified according to a unique identification number and its herd code number. This allowed identification and recorded follow-up of health history, reproduction, milk production, activity, milk conductivity, and other parameters through peripheral and central databases.
      None of the participating farms performed any special preparations for the drying period, such as gradual or intermittent milking or a change in food ration. All cows were generally dried off with antibiotic intramammary treatment after their last milking before entering the dry period. Two farms (Darom and Tzvaim) used Fatroximin dry tubes (rifaximin 0.1 g, ATI, Fatro S.p.A., Italy), and the other farms used Nafpenzal DC [procaine benzyl penicillin, 300 mg; dihydrostreptomycin (as the sulfate), 100 mg; and nafcillin (as the sodium salt), 100 mg; Intervet International B.V.]. Additional procedures, such as internal teat sealing, were not practiced on any of the farms. During the study period, antibiotic DCT was not used on the cows participating in the trial.
      The dry-off routine on all farms was based on drying off the cows approximately 60 d before the expected calving date. Dry cows were kept in 2 groups: a far-off dry group (from 60 to 21 d before expected calving) and a close-up dry group (from 20 d before expected calving until calving). Cows were shifted to the close-up group based on their BCS. Roughly, cows with a BCS <3.0 were shifted 3 wk before the expected calving day, whereas cows with higher BCS ≥3.0 were shifted 2 wk before the expected calving day.
      Nutrition for the dry cows during the far-off period was based on a dry ration that contained 12% CP, 1.4 Mcal of NEL/kg of DM, and 75% roughage; for the close-up period, the ration contained 14.5% CP, 1.55 Mcal of NEL/kg of DM, and 60% roughage. The main roughage sources used for both rations were wheat hay and wheat silage. The rations were formulated to meet
      • National Research Council
      Nutrient Requirements of Dairy Cattle.
      recommendations for vitamins A, D, and E. The contents of Ca and P in the dry period rations were 0.39 and 0.32%, respectively.

      Preparation of Experimental and Control Solutions and Treatment

      Saline solution (Teva Medical Ltd.) intended for the control group was prepared under aseptic conditions (Biological Industries). The bCNH was produced according to Bradford protein assay by enzymatic hydrolysis of bovine CN under aseptic conditions (Biological Industries). The solution was a pyrogen-free liquid preparation, supplied in sterile vials. The endotoxin level in the working solution was <1.0 endotoxin units/mL, according to limulus amebocyte lysate test. The concentration of the final solution was 60 mg diluted in 20 mL of sterile solution (i.e., 3 mg/mL).
      Immediately after mammary-secretion collection on the treatment day (d 0), 20 mL of either bCNH or saline solution was infused into the teats. In the first 2 wk posttreatment, each treated cow underwent 5 clinical observations (posttreatment d 1, 2, 3, 7, 14) by a clinical technician for assessment of teat morphology, teat leakage, and the cow's general status. Aseptic quarter milk samples were delivered to the Laboratory of Udder Health and Milk Quality (Israel Dairy Board, The National Service for Udder Health and Milk Quality, Caesaria Industrial Park, Israel).

      Other Study Procedures

      The farms were intentionally recruited at intervals of 1 to 2 wk, to give all farm personnel the necessary attention during the first weeks of the trial. Before recruitment, all farm personnel participated in 2 instruction sessions, given by the same instructor with the same guidelines. Instruction included an explanation of the theory behind the written guidelines and standard operating procedures, trained to aseptically collect milk samples and to use a standardized mastitis severity scoring system, and a practical demonstration of the proper way to treat and sample udder quarters.
      Sampling of mammary secretions from each udder quarter was performed aseptically according to guidelines adapted from the US
      • National Mastitis Council
      Laboratory and Field Handbook on Bovine Mastitis.
      . A few streams of foremilk were discarded before filling the sterile sample tubes with 5- to 10-mL squirts of milk.

      Bacteriological Diagnosis of Milk Samples

      Examination of bacterial growth and diagnostics followed the official Israeli procedures of the Israel Dairy Board, the Laboratory Handbook on Bovine Mastitis of the National Mastitis Council (
      • Hogan J.S.
      • González R.N.
      • Harmon R.J.
      • Nickerson S.C.
      • Oliver S.P.
      • Pankey J.W.
      • Smith K.L.
      • Hogan J.
      • Armas-Portela R.
      • Harmon R.
      • Nickerson S.C.
      • Oliver S.
      • Pankey J.
      Laboratory Handbook on Bovine Mastitis.
      ), the guidelines of the Microbiological Procedures for the Diagnosis of Bovine Udder Infections and Determination of Milk Quality of the
      • National Mastitis Council
      Microbiological Procedures for Use in the Diagnosis of Bovine Udder Infection and Determination of Milk Quality.
      , and Bergey's Manual (
      • Garrity G.M.
      • Brenner D.J.
      • Krieg N.R.
      • Staley J.T.
      Bergey's Manual of Systematic Bacteriology – Vol. 2: The Proteobacteria.
      ). The laboratory works under IEC17025 International Organization for Standardization (ISO) standards.
      The samples were analyzed for bacterial growth from 0.01 mL of milk spread on a blood agar plate (tryptic soy agar + 5% sheep blood). The plates were sectioned into 4 quadrants and incubated for 48 to 72 h at 36°C (±1°C). Typical coliform colonies were specifically identified by colony morphology, indole test, inoculation onto selective media—MacConkey agar and eosin methylene blue, and negative oxidase test; other tests, such as citrate, ornithine, and motility tests were performed for final identification of species, including Escherichia coli, Klebsiella, Enterobacter, and Serratia. Typical Staphylococcus spp. colonies were identified by colony morphology, hemolysis, positive catalase test, and Staphylase test. When a mismatch occurred between the morphology and the results of the Staphylase test, a tube coagulase test was performed. Typical Streptococcus spp. colonies were specifically identified by colony morphology; type of hemolysis; negative catalase test; positive Gram stain; Christie–Atkins–Munch-Petersen test; a serological test for grouping according to Lancefield's system into A, B, C, D, and G; esculin reaction; and a hippurate test for final identification of species, including Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Streptococcus canis. Typical Pseudomonas colonies were identified by colony morphology, hemolysis, characteristic fruit-like odor, and positive oxidase test. Transfer to MacConkey agar was performed in the case of questionable Aeromonas growth. Yeast and mold were identified by colony morphology and specific microscopic morphology. Corynebacterium bovis was identified mainly by colony morphology on blood agar. In general, a level of ≥1 colony/0.01 mL of major udder pathogens and ≥1 colonies/0.01 mL of minor udder pathogens in pure culture were designated as positive (
      • National Mastitis Council
      Laboratory and Field Handbook on Bovine Mastitis.
      ;
      • Torres A.H.
      • Rajala-Schultz P.J.
      • DeGraves F.J.
      Diagnosis of intramammary infections at dry-off based on sampling strategy, epidemiology of pathogens, and agreement beyond chance.
      ).
      Bacteriological samples from each quarter were collected aseptically 4 times: (1) 2 d before the time of treatment administration; (2) at the time of treatment administration (d = 0); (3) 3 d after calving; and (4) 5 d after calving. Bacterial types isolated from each sampling time are listed in Table 2, Table 3 (pretreatment: 2 d and d = 0, respectively) and Table 4, Table 5 (3 d and 5 d postcalving, respectively).
      Table 2Bacterial types isolated from the participating quarters in the first pretreatment milk sampling, by treatment group [number of cows and quarters, respectively: control: 67, 268; bovine casein hydrolysate (bCNH): 100, 397]
      GroupInitial bacterial isolation 2 d before treatment
      C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, Str = Streptococcus spp., E. coli = Escherichia coli, Strmyc = Streptomyces spp., Prot = Prototheca spp., Minor path. = Corynebacteriumspp., Streptococcusspp., “+” indicates that 2 different bacteria were isolated.
      UninfectedCNSC. bov.BacillC. jac.StrE. coliStrmycProtCNS + Minor path.Total quarters
      Control2114253220111268
      78.7315.671.871.120.720.7500.370.370.37
      bCNH31467346102397
      79.1016.880.761.011.510.2500.5
      Total52510987821113665
      78.9416.391.201.051.200.300.150.150.150.45100
      1 C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, Str = Streptococcus spp., E. coli = Escherichia coli, Strmyc = Streptomyces spp., Prot = Prototheca spp., Minor path. = Corynebacteriumspp., Streptococcusspp., “+” indicates that 2 different bacteria were isolated.
      Table 3Bacterial types isolated from the participating quarters in the second pretreatment milk sampling (treatment day), by treatment group [number of cows and quarters, respectively: control: 69, 273; bovine casein hydrolysate (bCNH): 98, 392]
      GroupInitial bacterial isolation on day of treatment (0 d)
      C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, Str = Streptococcus spp., Strmyc = Streptomyces spp., Minor path. = minor pathogens (Streptococcusspp., Corynebacteriumspp., Coliforms), “+” indicates that 2 different bacteria were isolated.
      UninfectedCNSC. bov.BacillC. jac.StrStrmycCNS + Minor path.Total
      Control21743334111273
      79.4915.751.101.101.470.370.370.36
      bCNH30372247013392
      77.2918.370.511.021.7900.260.77
      Total5201155711124665
      78.1917.290.751.051.650.150.300.60100
      1 C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, Str = Streptococcus spp., Strmyc = Streptomyces spp., Minor path. = minor pathogens (Streptococcusspp., Corynebacteriumspp., Coliforms), “+” indicates that 2 different bacteria were isolated.
      Table 4Bacterial types isolated from the participating quarters in the first postcalving milk sampling (3 d postcalving), by treatment group [number of cows and quarters, respectively: control: 66, 262; bovine casein hydrolysate (bCNH): 90, 357]
      GroupBacterial isolation 3 d postcalving
      C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, S. aur. = Staphylococcus aureus, Str = Streptococcus spp., E. coli = Escherichia coli, St. dys. = Streptococcus dysgalactiae, Minor path. = minor pathogens (Corynebacteriumspp., Streptococcusspp., Proteus, Streptococcus fecum), Miss. = missing, “+” indicates that 2 different bacteria were isolated.
      UninfectedCNSC. bov.BacillC. jac.S. aur.StrE. coliSt. dys.CNS + Minor path.Miss.Total
      Control131865403355155262
      5032.821.911.531.151.151.911.915.731.91
      bCNH2168749131147114357
      60.5124.371.122.520.280.840.283.921.963.081.12
      Total2591739131641912269619
      56.0627.951.452.100.160.970.653.071.944.21.45100
      1 C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, S. aur. = Staphylococcus aureus, Str = Streptococcus spp., E. coli = Escherichia coli, St. dys. = Streptococcus dysgalactiae, Minor path. = minor pathogens (Corynebacteriumspp., Streptococcusspp., Proteus, Streptococcus fecum), Miss. = missing, “+” indicates that 2 different bacteria were isolated.
      Table 5Bacterial types isolated from the participating quarters in the second postcalving milk sampling (5 d postcalving), by treatment group [number of cows and quarters, respectively: control: 66, 261; bovine casein hydrolysate (bCNH): 91, 361]
      GroupBacterial isolation 5 d postcalving
      C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, S. aur. = Staphylococcus aureus, Str = Streptococcus spp., E. coli = Escherichia coli, Strmyc = Streptomyces spp., St. dys. = Streptococcus dysgalactiae, Str. uberis = Streptococcus uberis, Others = Prototheca, coliforms, Streptococcus fecum, Arcanobacter pyogenes, Bacillus cereus, Enterobacter sakazii, Minor path. = minor pathogens (Streptococcusspp., Prototheca, Streptococcus uberis, Proteus, Streptococcus fecum, Arcanobacter pyogenes), Miss. = missing, “+” indicates that 2 different bacteria were isolated.
      UninfectedCNSC. bov.BacillC. jac.S. aur.StrE. coliStrmycSt. dys.OthersCNS + Minor path.Str. + Str. uberisMiss.Total
      Control13188802445145414261
      50.1933.723.070.771.531.531.920.381.531.921.530.381.53
      bCNH231867313010048404361
      6423.821.940.830.280.8302.7701.112.221.1101.11
      Total277174153374151813818622
      58.1827.972.410.481.130.642.410.161.291.232.091.290.161.29100
      1 C. bov. = Corynebacterium bovis, Bacill = Bacillus spp., C. jac. = Corynebacterium jacom, S. aur. = Staphylococcus aureus, Str = Streptococcus spp., E. coli = Escherichia coli, Strmyc = Streptomyces spp., St. dys. = Streptococcus dysgalactiae, Str. uberis = Streptococcus uberis, Others = Prototheca, coliforms, Streptococcus fecum, Arcanobacter pyogenes, Bacillus cereus, Enterobacter sakazii, Minor path. = minor pathogens (Streptococcusspp., Prototheca, Streptococcus uberis, Proteus, Streptococcus fecum, Arcanobacter pyogenes), Miss. = missing, “+” indicates that 2 different bacteria were isolated.

      Case Definition and Analysis of Prevention and Cure of IMI

      To be eligible for inclusion in the analysis of the prevention rate of IMI, a quarter had to be free of pathogens in the 2 successive milk samples before dry-off. A quarter was defined as free of new IMI (or prevention) if both samples, taken after calving, were pathogen free. For the analysis of curing an existing IMI caused by CNS, the infection status before dry-off of a quarter was identified as infected if CNS were diagnosed in 1 of 2 pretreatment samples. Cure was then defined when CNS were not diagnosed in either of the 2 postcalving samples. An udder quarter was considered cured of CNS if they were not detected in both samples (taken 3 and 5 d postcalving). Udder quarters that cultured positive for CNS before treatment but positive to other pathogens postcalving were considered cured of CNS. Analysis was conducted by chi-squared test and followed by a general linear mixed model as described in the Statistical Analysis section. A sample was considered contaminated when 3 or more dissimilar colony types were isolated. Contaminated quarters were omitted from the analyses of prevention and cure rates.
      A quarter was defined as noninfected if bacteria were not detected. The final database included 166 quarters (67 for the control group and 99 for the bCNH group), among them 27 quarters that were detected with a new pathogen after calving. The predominant bacteria isolated in both groups before dry-off were CNS, accounting for 72% of all isolated bacteria.
      Clinical assessment of the quarters was performed by the clinical investigator on d 0, 1, 2, 3, 7, and 14 after treatment and calving. The following index was used: 0 = normal, 1 = red, slight swelling, and 2 = obvious, severe swelling. Teat leakage was also assessed, for a few seconds each time, according to the index: 0 = normal (i.e., no leakage), 1 = occasional leakage (i.e., not constant leakage), and 2 = constant leakage (i.e., constant flow of milk). Cows with severe clinical mastitis before eligibility (abnormal milk accompanied by systemic signs) were not eligible and were treated immediately according to existing farm protocols.

      Determination of Minimal Required Sample Size

      Sample size was determined at the quarter level. The primary pivotal variable was the cure rate of subclinical IMI. The null hypothesis was that the cure rate of IMI by bCNH would not differ significantly from that achieved for a negative control; therefore, a 2-sided tailed test was used. Assumptions were a bCNH cure rate of 70% and a negative control cure rate of 30% (
      • Soback S.
      • Ziv G.
      • Winkler M.
      • Saran A.
      Systemic dry cow therapy.
      ) postcalving. Based on these rates, a 2-sided chi-squared test, α = 0.05, and 90% power, the minimum required sample size was 31 quarters per treatment (PROC POWER, SAS). Because the bacterial status of a quarter was unknown at the moment of randomization, and assuming that approximately 30% of the quarters would be infected at dry-off, 31 × 100/30 = 104 quarters were needed per treatment group. Considering dropout due to animal death and other potential events, sample size was increased to 116 quarters (29 cows) per treatment group. However, due to the requirements of the registration authorities, a minimum of 100 bCNH-treated cows was set for safety evaluation. Finally, the sample size was set at 170 cows with a randomization ratio of 10 bCNH-treated cows to 7 negative control cows (i.e., 100 cows in the bCNH group and 70 cows in the negative control group).

      Postcalving Follow-Up

      Milk yield was routinely measured by milk meters at every milking, and the measurements were combined for daily milk yield, then automatically recorded in the farm's herd-management software and in NOA as described above. The data were then collected and mean milk yield for each month in milk (MIM), out of 10, was calculated. Data of milk SCC, and protein and butterfat contents for each of the participating cows were collected for the whole lactation through routine monthly milk sampling (i.e., test-day). Analysis of SCC was carried out at the cow level and based on test-day data from the first 10 mo of milk recordings postcalving. To achieve a more normal distribution, each SCC measurement was first transformed to the log scale. Calving diseases were collected from the ICBA herdbook. Calving diseases included in the analysis were stillbirth (dead calf within 24 h of birth), retained placenta (fetal membranes visible in vulva >24 h postpartum), endometritis (abnormal vaginal discharge at routine postpartum examination), and ketosis (urine acetoacetic acid concentration ≥1.5 mmol/L at routine postpartum examination). The risk of being culled ≤60 d postpartum was also included.

      Statistical Analysis

      Version 9.2 of SAS was used for all analyses. In general, effects were considered significant if P < 0.05. In the univariable analysis, proportions were compared using Pearson's chi-squared test with Yates's correction for continuity.
      Multivariable analyses were conducted using generalized linear mixed models (PROC GLIMMIX, SAS Institute, 2006, and R with binominal distribution and logit as Link function) to study the effects of treatment (fixed effect with 2 levels: bCNH or control) alongside the independent variables: farm (random effect with 4 levels), parity group (fixed effect with 2 levels: primiparous or multiparous), and averages of milk yield and SCC level of previous lactation, on the odds of either cure or prevention of IMI. Two-way interactions (herd × treatment, and parity × treatment) were also tested. The differences between the categories were tested using the PDIFF option (SAS).
      Average daily milk yield for 305 d was modeled at the cow level using daily milk production data, which were categorized to 10 MIM and adjusted to average daily milk yield for 305 d and average of monthly SCC level for 305 d of previous lactation. The other independent variables included in the linear mixed model were farm (as random effect with 4 levels), parity group (fixed effect with 2 levels: second and ≥third), and treatment groups (fixed effects with 2 levels: bCNH and control). Two-way interactions (parity × treatment and MIM × treatment) were also tested. The differences between the categories were tested using the PDIFF option.
      Log-transformed SCC was adjusted to the average log-transformed SCC in the previous lactation (305 d). The other independent variables in the linear mixed model were farm (as a random effect), parity, treatment, test-day, and the 2-way interactions (parity × treatment, and test-day × treatment) were also tested. The differences between the categories were tested using the PDIFF option. In a similar model, the relationship between milk yield and SCC as a categorial variable was analyzed. The SCC was categorized to 4 levels: (1) <100,000; (2) 101,000–200,000; (3) 201,000–400,000, and (4) >400,000. The other independent variables were as mentioned above. Neither parity nor average SCC of previous lactation had a significant effect, and therefore were excluded from the model.

      RESULTS

      Protocol Deviations

      Several minor deviations from the protocol occurred during the study (Figure 1). On one of the farms, 7 cows were assessed for eligibility, enrolled in the study, but not randomized and not allocated to the destined treatment group. On another farm, the technician forgot to sample 2 cows (1 from each treatment group) on both posttreatment sampling days. We have no reason to believe that these minor deviations had any effect on the study results.

      Analysis of Prevention of New IMI

      Of the 644 quarters available for inclusion in the study, 461 (71.6%) were not infected at dry-off and were eligible for the analysis of prevention of new IMI. Of these, 4 had contaminated samples postpartum and could not be included in the analysis. Of the remaining 457 quarters, bacteriological results were obtained from 452 quarters at the first sampling 3 d after calving, and from 453 quarters 5 d after calving. The distribution of quarters for each group (control and bCNH) within each farm that participated in the analysis of prevention of new IMI was between 20% and 30%.
      The proportion of quarters without IMI postcalving was 59.2% and 45.6% in the bCNH group and control group, respectively (P = 0.006). After adjusting for parity group, averages of milk yield, and SCC in previous lactation in the multivariable analysis (Table 6), the odds for preventing new IMI were 2.15 times higher for cows in the bCNH group compared with those in the control group. The odds of preventing new IMI in primiparous cows were 1.36 times higher than for multiparous cows (Table 6), but the interaction of parity and treatment was not statistically significant and was therefore removed from the model.
      Table 6Effect of parity, treatment, and means of milk yield and SCC of previous lactation on prevention rate, and the odds of preventing new IMI of parity and treatment, obtained from a generalized linear mixed model
      VariableEstimateSEP > |z|Odds ratioCI (95%)
      Intercept−0.8770.8490.301
      Milk0.0030.0250.903
      SCC0.00050.00090.581
      Parity (2 vs. 1)0.3110.1520.041.361.01–1.84
      Treatment (bCNH
      bCNH = bovine casein hydrolysate.
      vs. control)
      −0.7680.310.0132.151.15–4.00
      1 bCNH = bovine casein hydrolysate.

      Analysis of Prevention of New IMI Caused by CNS

      The proportion of quarters acquiring new IMI caused by CNS was significantly different between bCNH-treated and control groups (P = 0.007). The percentage of new IMI caused by CNS in bCNH and control groups was 13% and 23%, respectively. The odds of a quarter receiving bCNH at dry-off being infected with CNS were reduced by a factor of 2.20 (P < 0.020). The available sample size and the frequency of the other intramammary pathogens found in the study did not enable an analysis with sufficient power for any of the other pathogens.

      Analysis of Cure Rate of IMI Present at Dry-Off

      Coagulase-negative staphylococci were the dominant bacteria in samples taken 2 d pretreatment and on day of treatment (78% and 79.3%, respectively). Other bacteria in both pretreatment bacteriological samples were (Table 2, Table 3): (1) environmental pathogens: Streptococcus spp., E. coli, Bacillus spp., and Proteus spp. and (2) commensal pathogens: Corynebacterium bovis. The prevalence for environmental pathogens was 1.76%, and for the commensal pathogens 4.6%. Coagulase-negative staphylococci were also dominant in samples taken 3 and 5 d postcalving (66% and 73%, respectively). Other bacteria in both postcalving bacteriological samples were (Table 4, Table 5): (1) contagious pathogens: Staphylococcus aureus; (2) environmental pathogens: E. coli, Enterobacter spp., Streptococcus uberis, Streptococcus dysgalactiae, Klebsiella spp., Bacillus spp., Proteus spp., and Prototheca; and (3) commensal pathogens: Corynebacterium bovis, CNS, and Trueperella pyogenes (Arcanobacterium pyogenes). However, all commensal pathogens can also be defined as pathogens causing IMI. The prevalence for contagious pathogens was 2.5%, for environmental pathogens 5.5%, and for the commensal pathogens (without CNS) 4.6%. Thus, the available sample size and frequency of those other intramammary pathogens did not enable an analysis with sufficient power for any of the other pathogens.
      For the analysis of curing an existing IMI caused by CNS, the infection status of a quarter before dry-off was defined as infected if (1) CNS were diagnosed in 1 of 2 pretreatment samples; (2) CNS were diagnosed in both pretreatment samples; (3) CNS were diagnosed only in the second bacteriological sample taken on day of treatment. Cure was then defined when CNS were not diagnosed in either of the 2 postcalving samples. The odds of being cure was consistently higher in bCNH-treated quarters compared with control quarters under all 3 definitions, and these ratios were similar to each other: in (1) 4.80 (95% CI: 0.75 to 30.75, P = 0.09); in (2) 2.93 (95% CI: 0.32 to 26.36, P = 0.32); and in (3) 4.94 (95% CI: 0.90 to 26.54, P = 0.06). Cure rate of CNS was also similar in bCNH-treated cows: 42%, 35%, and 40% for definitions (1), (2), and (3), with 25%, 27%, and 25% of the controls, respectively. Alternative (1) was then further used to examine the effect of bCNH on curing IMI caused by CNS.
      Cure percentage differed between the 2 groups (25% and 42% for control and bCNH, respectively, P = 0.05). In the multivariable analysis, which was adjusted to parity group, farm, and averages of milk yield and monthly SCC of previous lactation, we found that the odds of having a cured IMI caused by CNS in quarters treated with bCNH were 4.80 times higher (95% CI: 0.75 to 30.75, P = 0.09) than in those treated with saline.

      SCC, Milk Components, and Milk Production for 305 d Postcalving

      SCC

      Log SCC of cows in the control group was greater than in the bCNH group (P = 0.008) after adjusting for parity group, mean log of the month SCC values of previous lactation, test-day, and random farm effect. The least squares means estimates of the bCNH group were lower in 6 of the 10 MIM (P < 0.05) compared with the control group (Figure 2). This result is important because log SCC levels of cows from both groups were not significantly different along the previous lactation (Table 7).
      Figure thumbnail gr2
      Figure 2Least squares means ± SE of log-transformed SCC of cows treated with bovine casein hydrolysate (bCNH) at dry-off and those in the negative control group by month in the subsequent lactation, adjusted for parity group and farm, and the average log-transformed SCC of the lactation before dry-off. *P < 0.05.
      Table 7Least squares means of log SCC on all test-days of the lactation preceding recruitment, in the control and bovine casein hydrolysate (bCNH) groups; differences, F-ratio, and P-values are included
      Test- dayControlbCNHDifferenceF-ratioP-value
      LSMSELSMSE
      15.0320.0505.0070.0420.0240.1330.715
      24.8890.0494.8860.0410.0200.1000.751
      34.9170.0494.8520.0420.0650.9960.318
      44.9120.0494.8200.0420.0922.0100.156
      54.9180.0494.9130.0410.0040.0050.941
      64.9210.0504.9170.0410.0040.0040.949
      74.9520.0494.9420.0410.0100.0040.867
      84.9710.0504.9170.0420.0540.0280.410
      95.0060.0495.0240.042−0.0170.0730.787
      104.9880.0525.0090.044−0.0200.0890.764

      Milk Production

      Milk production, adjusted to average daily milk yield and average monthly SCC for 305 d in the previous lactation, was significantly higher (by 2.1 kg/d) for the bCNH group than for the control group for the 305-d period (P < 0.007). Milk production by the bCNH group was significantly higher than by the control group for the first to tenth test-days (Figure 3). In a linear mixed model in which 4 SCC levels were categorized (as fixed effects), milk production was negatively affected by increasing level of SCC (Table 8). When adjusting for all other variables in the model, the daily milk production of cows in the bCNH group was similar to the result of the model without SCC. This finding is remarkable for its independence of the effect of SCC (Table 8), meaning that the increase in milk yield in the bCNH group can also be attributed to the treatment.
      Figure thumbnail gr3
      Figure 3Least squares means ± SE of milk yield of cows treated with bovine casein hydrolysate (bCNH) at dry-off and those in the negative control group by month in the subsequent lactation, adjusted for parity group, farm, and the average milk yield of the lactation before dry-off. *P < 0.05; **P < 0.01.
      Table 8Associations between daily milk production (kg) and SCC on the 10 test-days of the lactation postcalving, estimated with a mixed model with repeated measurements and a random farm
      ParameterVariableEstimateSEP > |t|
      Intercept18.833.144<0.0001
      Milk (mean of previous lactation)0.53080.0083<0.0001
      Month in milk
      1−0.85910.42380.043
      23.88120.7129<.0001
      30.58580.66850.376
      40.76380.66520.235
      51.95680.65980.003
      6−1.7470.67780.009
      7−2.1540.74040.004
      8−0.1911.06160.856
      9−3.4921.51510.021
      100
      Treatment group
      Control−1.720.8430.043
      bCNH
      bCNH = bovine casein hydrolysate.
      0
      SCC level (cells/mL)
      <100,0003.910.718<0.0001
      100,000–200,0003.490.727<0.0001
      200,000–400,0001.110.8400.002
      >400,0000
      1 bCNH = bovine casein hydrolysate.

      Milk Fat and Protein

      Association between daily fat percentage and protein percentage (as dependent variables) and MIM, SCC level, and bCNH and control groups reveals that neither fat nor protein were affected by the bCNH treatment (P > 0.675 and P > 0.28 for fat and protein, respectively).

      Adverse Events

      Ten adverse events occurred during the trial period. None of them were categorized as “probably drug-related” (i.e., adverse events were not thought to be drug-related in any case). At most, adverse events were categorized as “possibly drug-related” if such a relation could not be ruled out. The most common adverse event was abortion, with 6 cases occurring during the trial period, 4 in the bCNH (4/170) group and 2 in the control group (2/100). Of these, 4 cases (3 in the bCNH group and 1 in the control group) involved the abortion of twins. However, the ratio of abortions in our trial did not exceed the normal annual ratio on Israeli farms. In addition, abortion risk is normally higher with twins compared with normal calving.

      DISCUSSION

      The present study had 2 primary objectives: (1) to investigate whether intramammary infusion of one dose of bCNH at dry-off would prevent new IMI during the dry period, in light of the well-documented evidence of a high risk for new IMI in the initial phase of dry-off and during the calving period (
      • Ruegg P.L.
      A 100-year review: Mastitis detection, management, and prevention.
      ;
      • Sordillo L.M.
      Mammary gland immunology and resistance to mastitis.
      ), and (2) to determine whether bCNH infusion at dry-off can cure existing IMI. The secondary objectives were to examine the effect of bCNH treatment on milk yield, milk composition, and SCC for 305 d postcalving.
      The dry period begins with cessation of milking, either abrupt or gradual. This results in milk stasis in the udder, which leads to udder engorgement and tight junction leakage, and triggers involution. The process of active involution in goats and cows has been shown to be triggered by the milk plasmin system, which liberates active peptide from the N-terminal part of β-CN (
      • Silanikove N.
      • Iscovich J.
      • Leitner G.
      Therapeutic treatment with casein hydrolyzate eradicates effectively bacterial infection in treated mammary quarters in cows.
      ). Plasmin in milk is found mostly in its inactive form, plasminogen, and the conversion of plasminogen to plasmin is modulated by plasminogen activators (
      • Politis I.
      Plasminogen activator system: implications for mammary cell growth and involution.
      ;
      • Silanikove N.
      • Shamay A.
      • Shinder D.
      • Moran A.
      Stress down regulates milk yield in cows by plasmin induced β-casein product that blocks K+ channels on the apical membranes.
      ). Within 13 d of cessation of milking, plasmin activity in dairy cow mammary secretions increases substantially (
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ).
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      showed that the infusion of bCNH, which contains active β-CN-derived peptide, enhances the immune process, and dramatically accelerates the rate of involution in goats and cows: it was completed within 3 d to 5 d, as compared with the natural involution process which took 14 d to 21 d (
      • Holst B.D.
      • Hurley W.L.
      • Nelson D.R.
      Involution of the bovine mammary gland: histological and ultrastructural changes.
      ;
      • Capuco A.V.
      • Akers R.M.
      Mammary involution in dairy animals.
      ;
      • Shamay A.
      • Shapiro F.
      • Mabjeesh S.J.
      • Silanikove N.
      Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.
      ). Infusion of bCNH has also been associated with earlier increases in the concentrations of components of the innate immune system: lactoferrin (antimicrobial protein) and IgG (
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ).
      The immunomodulatory effect of infusion of bCNH into the mammary gland has been investigated in several studies (
      • Shamay A.
      • Shapiro F.
      • Mabjeesh S.J.
      • Silanikove N.
      Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.
      ;
      • Silanikove N.
      • Iscovich J.
      • Leitner G.
      Therapeutic treatment with casein hydrolyzate eradicates effectively bacterial infection in treated mammary quarters in cows.
      ,
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ;
      • Leitner G.
      • Jacoby S.
      • Maltz E.
      • Silanikove N.
      Casein hydrolyzate intramammary treatment improves the comfort behavior of cows induced into dry-off.
      ;
      • Ponchon B.
      • Lacasse P.
      • Silanikove N.
      • Ollier S.
      • Zhao X.
      Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution.
      ). Most of these researchers investigated the mechanism involved following administration of bCNH, via either a single infusion or multiple infusions over several days (
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ;
      • Ponchon B.
      • Lacasse P.
      • Silanikove N.
      • Ollier S.
      • Zhao X.
      Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis (beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution.
      ;
      • Britten J.
      • Rood K.
      • Wilson D.
      Intramammary infusion of casein hydrolysate for involution of single mastitic mammary quarters elevating cow-level somatic cell count.
      ). A single bCNH infusion into the mammary gland of milking goats while still milking the treated glands had a transient effect on Na+, plasmin, and plasmin activator by 12 h posttreatment, which clearly indicated disruption of the tight junctions very soon after the infusion (
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ). The effect of a single bCNH infusion was also tested in combination with DCT (
      • Leitner G.
      • Jacoby S.
      • Silanikove N.
      An evaluation of casein hyrolyzate in combination with antibiotic for bacterial cure and subsequent increase in milk yield in dairy cows.
      ), and as an alternative to antibiotic dry therapy (
      • Britten J.
      • Rood K.
      • Wilson D.
      Intramammary infusion of casein hydrolysate for involution of single mastitic mammary quarters elevating cow-level somatic cell count.
      ;
      • Britten J.E.
      Evaluation of casein hydrolysate as an alternative dry-off treatment and milk quality management tool in dairy cows.
      ). Although involution occurred in the latter according to the biomarkers, the cure rate was difficult to evaluate due to the small number of quarters involved (7 quarters allocated for 4 treatments). Our study is therefore the first large study investigating the efficacy of bCNH, administered as a single dose, as a DCT (i.e., without antibiotic), in preventing new IMI and curing existing ones.
      The prevalence of positive bacterial cultures for CNS (i.e., without contaminated bacteria) from milk samples before treatment was similar in both groups. Postcalving, there were more positive bacterial cultures at the udder quarter level in control versus treated udder quarters. The 10% difference was in the medium value range of a meta-analysis performed by
      • Robert A.
      • Seegers H.
      • Bareille N.
      Incidence of intramammary infections during the dry period without or with antibiotic treatment in dairy cows—A quantitative analysis of published data.
      showing a −2.1% to +22.0% difference in dairy cows that received antibiotic DCT. The results for new CNS cases postcalving were even more salient compared with previous studies, with a difference of 12.0%. These results can be compared with the meta-analysis that reported a difference range of only −8.5 to +5.5% with “a limited magnitude or not effective prevention” of CNS in cows treated with antibiotic DCT (
      • Robert A.
      • Seegers H.
      • Bareille N.
      Incidence of intramammary infections during the dry period without or with antibiotic treatment in dairy cows—A quantitative analysis of published data.
      ). The significantly lower rate of infection in the treated udder quarters, for CNS, highlights the importance of bCNH in the clearance of infections during the early dry period. It is interesting to note that the high prevalence of CNS in the glands before dry-off as found in our trial is similar to research done in Israel (
      • Leitner G.
      • Jacoby S.
      • Silanikove N.
      An evaluation of casein hyrolyzate in combination with antibiotic for bacterial cure and subsequent increase in milk yield in dairy cows.
      ) and abroad (
      • Vanderhaeghen W.
      • Piepers S.
      • Leroy F.
      • Van Coillie E.
      • Haesebrouck F.
      • De Vilegher S.
      Invited review: Effect, persistence, and virulence of coagulase-negative Staphylococcus species associated with ruminant udder health.
      ).
      Our results also highlight the efficacy of bCNH in curing cases of CNS. The cure rate in our study was comparable to previous reports demonstrating that mammary secretions from involuted glands are bactericidal and bacteriostatic against a wide range of pathogens (
      • Breau W.C.
      • Oliver S.P.
      Growth-inhibition of environmental mastitis pathogens during physiological transitions of the bovine mammary gland.
      ;
      • Bushe T.
      • Oliver S.P.
      Natural protective factors in bovine mammary secretions following different methods of milk cessation.
      ;
      • Oliver S.P.
      • Bushe T.
      Growth-inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: Relation to secretion composition.
      ). The components responsible for those characteristics are the immunomodulators lactoferrin and IgG. The bactericidal activity of mammary secretions from the glands treated with bCNH, which started as early as 8 h after treatment, is related to the accelerated release of NO2 (
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ), which is a powerful bactericide (
      • Nathan C.
      • Shiloh M.U.
      Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens.
      ). It is suggested that bCNH treatment induces the secretion of short-lived cytokines, which in turn activate a NO burst that exceeds the antioxidant capacity of the mammary secretions.
      The relative effect for curing CNS in
      • Halasa T.
      • Nielen M.
      • Whist A.C.
      • Østerås O.
      Meta-analysis of dry cow management for dairy cattle. Part 2. Cure of existing intramammary infections.
      meta-analysis had an odds ratio of 1.65 during the dry period up to 21 d postcalving. The spontaneous overall cure rate from the meta-analysis (
      • Halasa T.
      • Nielen M.
      • Whist A.C.
      • Østerås O.
      Meta-analysis of dry cow management for dairy cattle. Part 2. Cure of existing intramammary infections.
      ) averaged 46% (37 to 56%). However, results of the current study indicated that bCNH has a higher cure efficacy of CNS, as indicated by a higher cure rate compared with the control (a difference of 17%) and an odds ratio of 4.80 (P = 0.09), compared with the odds ratio of 1.65 as stated above. Nevertheless, despite the higher cure rate of other bacteria of udder quarters treated with bCNH compared with control, we were not able to analyze bCNH efficacy of curing IMI, caused by other bacteria than CNS, due to insufficient power for any of the other pathogens.
      The recommended gold standard (duplicated or successive samples in series) aims to increase specificity and reduce false-positive results (
      • Oliver S.P.
      • Gonzalez R.N.
      • Hogan J.S.
      • Jayarao B.M.
      • Owens W.E.
      Microbiological Procedures for the Diagnosis of Bovine Udder Infection and Determination of Milk Quality.
      ). However, in field trials, determination of prevalence of infection after treatment can be based on a single aseptically collected milk sample (
      • International Dairy Federation
      Bovine mastitis: Definition and guidelines for diagnosis. IDF Bulletin 211/1987.
      ;
      • Torres A.H.
      • Rajala-Schultz P.J.
      • DeGraves F.J.
      Diagnosis of intramammary infections at dry-off based on sampling strategy, epidemiology of pathogens, and agreement beyond chance.
      ) under the

      Food and Drug Administration. 1996. CVM GFI #49. Target Animal Safety and Drug Effectiveness Studies for Anti-Microbial Bovine Mastitis Products (Lactating and Non-Lactating Cow Products). Docket Number: FDA-1993-D-0285.

      guideline #49 concerning minor pathogen diagnosis. In field studies, single milk samples are generally collected under the assumption that the presence of misclassification bias will be equally distributed among the experimental groups. Furthermore, comparison of prevention and cure rates to a baseline level, such as a negative control (administration of saline solution), equalizes the detection bias and validates the comparison of prevention- and cure-related differences.
      Isolation of ≥1 colony/0.01 mL of major contagious pathogens fits the criterion of true agreement of sample pairs for the diagnosis of IMI from udder quarter milk samples collected from cows with clinical (
      • Hogan J.S.
      • Galton D.M.
      • Harmon R.J.
      • Nickerson S.C.
      • Oliver S.P.
      • Pankey J.W.
      Protocols for evaluating efficacy of postmilking teat dips.
      ) and subclinical (
      • Østerås O.
      • Sølverød L.
      • Reksen O.
      Milk culture results in a large Norwegian survey—effects of season, parity, days in milk, resistance, and clustering.
      ;
      • Tenhagen B.A.
      • Köster G.
      • Wallmann J.
      • Heuwieser W.
      Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany.
      ) mastitis. For nonmajor microorganisms, the cut-off value of 10 colonies/0.01 mL results in better agreement.
      In general, variability in risk of infection among dairy herds is an important factor in infection control; however, no clear conclusions regarding this factor can currently be drawn (
      • Green M.J.
      • Bradley A.J.
      • Medley G.F.
      • Browne W.J.
      Cow, farm, and management factors during the dry period that determine the rate of clinical mastitis after calving.
      ;
      • Huijps K.
      • Hogeveen H.
      Stochastic modeling to determine the economic effects of blanket, selective, and no dry cow therapy.
      ). The probability of a new dry period IMI is influenced by the rate of exposure to potential pathogens (e.g., from the environment), factors that affect an individual cow's susceptibility to infection, and the protective effectiveness of medical interventions such as DCT (
      • Robert A.
      • Seegers H.
      • Bareille N.
      Incidence of intramammary infections during the dry period without or with antibiotic treatment in dairy cows—A quantitative analysis of published data.
      ;
      • Winder C.B.
      • Sargeant J.M.
      • Hu D.
      • Wang C.
      • Kelton D.F.
      • Leblanc S.J.
      • Duffield T.F.
      • Glanville J.
      • Wood H.
      • Churchill K.J.
      • Dunn J.
      • Bergevin M.D.
      • Dawkins K.
      • Meadows S.
      • Deb B.
      • Reist M.
      • Moody C.
      • O'Connor A.M.
      Comparative efficacy of antimicrobial treatments in dairy cows at dry-off to prevent new intramammary infections during the dry period or clinical mastitis during early lactation: a systematic review and network meta-analysis.
      ). Differences between dairy herds, although not necessarily significant, were identified in the patterns of prevention and cure rates during our study. Such differences, and the reasons for them, have not been quantified in any of the various studies conducted in recent years (
      • Dingwell R.T.
      • Leslie K.E.
      • Schukken Y.H.
      • Sargeant J.M.
      • Timms L.L.
      • Duffield T.F.
      • Keefe G.P.
      • Kelton D.F.
      • Lissemore K.D.
      • Conklin J.
      Association of cow and quarter-level factors at drying-off with new intramammary infections during the dry period.
      ;
      • Borm A.A.
      • Fox L.K.
      • Leslie K.E.
      • Hogan J.S.
      • Andrew S.M.
      • Moyes K.M.
      • Oliver S.P.
      • Schukken Y.H.
      • Hancock D.D.
      • Gaskins C.T.
      • Owens W.E.
      • Norman C.
      Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers.
      ;
      • Green M.J.
      • Bradley A.J.
      • Medley G.F.
      • Browne W.J.
      Cow, farm, and management factors during the dry period that determine the rate of clinical mastitis after calving.
      ) and were beyond the scope of the current study.
      In the meta-analysis carried out by
      • Halasa T.
      • Østerås O.
      • Hogeveen H.
      • van Werven T.
      • Nielen M.
      Meta-analysis of dry cow management for dairy cattle. Part 1. Protection against new intramammary infections.
      , which was corrected for information bias, antibiotic DCT was shown to provide significant protection against new IMI by Streptococcus species during the first 21 d postcalving; conversely, no protection was demonstrated against IMI by Staphylococcus species or coliforms at the udder quarter level. The limited or nonexistent preventive effect against CNS infection in mammary glands suggests that the infection may occur very late in the dry period, just before calving, or early postcalving (
      • Matthews K.R.
      • Harmon R.J.
      • Langlois B.E.
      Prevalence of Staphylococcus species during the periparturient period in primiparous and multiparous cows.
      ). Although no specific pathogen was reported,
      • Ruegg P.L.
      A 100-year review: Mastitis detection, management, and prevention.
      stated in her review that previous researchers (
      • Smith K.L.
      • Todhunter D.A.
      • Schoenberger P.S.
      Environmental pathogens and intramammary infection during the dry period.
      ;
      • Oliver S.P.
      • Sordillo L.M.
      Udder health in the periparturient period.
      ) had claimed that antibiotic DCT is not able to prevent new IMI entirely during the periparturient period. As a result, at the end of the dry period, cases treated with antibiotic dry-off therapies and untreated dairy cows are at the same risk for new IMI (
      • Dingwell R.T.
      • Leslie K.E.
      • Schukken Y.H.
      • Sargeant J.M.
      • Timms L.L.
      • Duffield T.F.
      • Keefe G.P.
      • Kelton D.F.
      • Lissemore K.D.
      • Conklin J.
      Association of cow and quarter-level factors at drying-off with new intramammary infections during the dry period.
      ;
      • Pyörälä S.
      Mastitis in post-partum dairy cows.
      ). Administration of bCNH, which accelerates involution of the treated quarters (3 to 5 d compared with 21 to 28 d after antibiotic use), is more successful in preventing new IMI than DCT in the initial phase of the dry period (
      • Capuco A.V.
      • Akers R.M.
      Mammary involution in dairy animals.
      ;
      • Shamay A.
      • Shapiro F.
      • Mabjeesh S.J.
      • Silanikove N.
      Casein-derived phosphopeptides disrupt tight junction integrity, and precipitously dry up milk secretion in goats.
      ). Thus, the higher efficacy of bCNH relative to negative control at the 2 time points at which the risk of contracting new IMI is at its highest (i.e., the initial phase of dry-off and before calving,
      • Ruegg P.L.
      A 100-year review: Mastitis detection, management, and prevention.
      ;
      • Sordillo L.M.
      Mammary gland immunology and resistance to mastitis.
      ) supports its success as an alternative to antibiotic DCT.
      In our study, there was a significant decrease in the average composite milk SCC values during the 305 d after calving in cows treated with bCNH, adjusted to SCC of previous lactation. This decrease can be clearly attributed to bCNH treatment. It is consistent with the prevention rate outcome seen in the bCNH-treated group and might be a result of the enhancement of innate immune capability and enrichment of milk secretion with bactericidal radicals (
      • Nathan C.
      • Shiloh M.U.
      Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens.
      ;
      • Silanikove N.
      • Shapiro F.
      • Shamay A.
      • Leitner G.
      Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates.
      ).
      Analysis for 305 d postcalving revealed a significant increase in the daily milk production of dairy cows treated with bCNH compared with controls. This increase was associated with the outcome of the bCNH treatment at dry-off because milk yield was adjusted to that of the previous lactation. It should be noted that IMI, with or without major involvement of the mammary parenchyma, have been associated with marked losses in milk yield (
      • Gröhn Y.T.
      • Wilson D.J.
      • Gonzalez R.N.
      • Hertl J.A.
      • Schulte H.
      • Bennett G.
      • Schukken Y.H.
      Effect of pathogen-specific clinical mastitis on milk yield in dairy cows.
      ;
      • Bobbo T.
      • Ruegg P.L.
      • Stocco G.
      • Fiore E.
      • Gianesella M.
      • Morgante M.
      • Pasotto D.
      • Bittante G.
      • Cecchinato A.
      Association between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits in dairy cows.
      ;
      • Hadrich J.C.
      • Wolf C.A.
      • Lombard J.
      • Dolak T.M.
      Estimating milk yield and value losses from increased somatic cell count on US dairy farms.
      ). Additionally, it is well documented that milk yield is negatively correlated with SCC (
      • Archer S.C.
      • Mc Coy F.
      • Wapenaar W.
      • Green M.J.
      Association between somatic cell count during the first lactation and the cumulative milk yield of cows in Irish dairy herds.
      ;
      • Hadrich J.C.
      • Wolf C.A.
      • Lombard J.
      • Dolak T.M.
      Estimating milk yield and value losses from increased somatic cell count on US dairy farms.
      ;
      • Heikkilä A.-M.
      • Liski E.
      • Pyörälä S.
      • Taponen S.
      Pathogen-specific production losses in bovine mastitis.
      ;
      • Potter T.L.
      • Arndt C.
      • Hristov A.N.
      Short communication: Increased somatic cell count is associated with milk loss and reduced feed efficiency in lactating dairy cows.
      ). However, our analysis of 305 d postcalving revealed that higher milk production in the bCNH-treated cows is independent of SCC level (Table 8). Taking all results of this trial together, the bCNH treatment has a higher prevention rate of IMI, probably because of the reduced load of bacteria in the gland.
      Administration of bCNH has been found to accelerate involution to its completion within 3 to 5 d (
      • Shamay A.
      • Shapiro F.
      • Leitner G.
      • Silanikove N.
      Infusions of casein hydrolyzates into the mammary gland disrupt tight junction integrity and induce involution in cows.
      ), compared with 21 to 35 d for the natural involution process (
      • Capuco A.V.
      • Akers R.M.
      Mammary involution in dairy animals.
      ). Therefore, shorter dry period lengths in combination with bCNH administration should be examined.
      Our results suggest that bCNH treatment is a valid alternative to antibiotics at dry-off as outcomes from its high efficacy to prevent new IMI after calving, and to cure subclinical IMI, mainly caused by CNS. This is supported by higher milk yield and lower SCC along the subsequent lactation. However, it remains necessary to examine the cure of other subclinical IMI caused by other pathogens in future studies because of the low prevalence of those pathogens in our study.

      CONCLUSIONS

      A single intramammary administration of bCNH as DCT is associated with prevention of IMI and the cure of subclinical mastitis during the dry period, mainly that caused by CNS. In addition, at 305 d postcalving revealed a significant increase in the daily milk production (by 2.1 kg/d) and decrease of SCC (P = 0.008) of dairy cows treated with bCNH compared with controls. As such, bCNH is proposed as an effective and safe alternative to the management of current DCT. However, while the results of the current study demonstrate the high efficacy of bCNH, its implementation and use on commercial dairy farms will first require regulatory approval.

      ACKNOWLEDGMENTS

      This clinical study was partially supported by Intervet Inc. d/b/a Merck Animal Health and by the Israel–US Binational Industrial Research and Development Foundation (BIRD, Project No. 1186). We also thank Ben Lifshitz (DVM), Jose Iscovich, David Javier Iscovich, regional veterinarians of “Hachaklait” (Mutual Society for Cattle Insurance and Clinical Veterinary Services in Israel), and the dairy farms for their superb cooperation. The authors have not stated any conflicts of interest.

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