Abstract
This study aimed the efficacy of meloxicam (MX) in treating acute clinical mastitis (ACM) without systemic symptoms in Holstein cows by studying improvement in udder pain, changes in prostaglandin E2(PGE2) and bradykinin (BK) levels in the milk, and milk yield (MY) after healing. Forty-two cows with ACM were randomly assigned to the MX treatment group (T group; n=21) and the control group (C group; n=21). At onset of illness (day 0), the T group received a 0.5 mg/kg subcutaneous (SC) injection of MX whereas the C group received 15 mL SC of saline solution as a placebo. Udder tenderness (UT) was measured, and milk samples were collected on days 0–3. There was little change in the MY of the T group before and after healing, whereas MY in the C group was significantly lower than after healing. UT on day 3 in the T group was significantly lower than that in the C group. PGE2 levels significantly decreased from day 0 to day 3 in both groups. A significant negative correlation between PGE2 and linear score was observed on day 1 in the T group, but not in the C group. In ACM without systemic symptoms, the administration MX may be useful for restoring MY and reducing udder pain after healing.
Keywords: acute clinical mastitis, Holstein dairy cow, meloxicam, prostaglandin E2, udder pain
Loss of milk production because of mastitis significantly impacts farms’ profitability. Adriaens et al. estimated the milk loss due to mastitis based on the difference between the expected and actual production, reporting an absolute median milk loss per case of 101.5 kg for a fixed period from −5 days before to 30 days after treatment [1]. Mastitis can also cause pain and discomfort in affected cows and affect milk production after its resolution. Leslie et al. highlighted that reducing the perceived pain and discomfort associated with clinical mastitis is essential [8].
meloxicam (MX), a nonsteroidal anti-inflammatory drug, selectively inhibits cyclooxygenase-2 (COX-2) activity and has less adverse gastrointestinal effects than nonspecific COX inhibitors such as flunixin meglumine and ketoprofen [7]. Systemic symptomatic mastitis caused by bacterial infection releases lipopolysaccharides (LPS), induces fever, and results in levels of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) levels. These inflammatory mediators promote upregulation of COX expression. MX alleviates mastitis symptoms, pain, and distress by inhibiting COX-2 expression [7].
Several previous studies [7] have reported therapeutic efficacy when MX is administered for acute mastitis with systemic symptoms. However, there have been no reports on the therapeutic effects of MX when administered for the treatment of acute mastitis without systemic symptoms.
This study aimed to examine the beneficial effects of MX on milk yield (MY) recovery and livestock welfare. We investigated the improvement in udder pain, changes in prostaglandin E2 (PGE2) and bradykinin (BK) concentrations in milk, and recovery of MY after healing after administering MX to Holstein cows with acute clinical mastitis (ACM) without systemic symptoms.
MATERIALS AND METHODS
This study was approved by the Okayama University of Science Experimental Animal Committee (Approval No.: Jitsu 2022-045).
Farms
Dairy cows of the Holstein-Friesian breed with ACM from four dairy farms in the Toyo region, Ehime, Japan, were included in this study. Two of the farms had the cows housed in tie stalls, fed separately, and had independent air and milk transport machines with 46 and 47 cows, respectively. One farm had a forestall, Partly Mixed Ration (PMR) feeding, and, two automatic milking systems, with 87 cows being milked. The last farm had a free barn, Total Mixed Ration (TMR) feeding, and a walk-through milking parlor, with 82 milked cows.
Cows
Forty-two cows with ACM in only one quarter without systemic symptoms, such as fever (above 39.5°C), depression, anorexia, cessation of peristalsis of the rumen, or diarrhea, were included in the study after rectal temperature measurement and examination at the onset of illness (day 0). The definition of symptoms of ACM-affected quarters was defined as heat, swelling, induration, and pain in the udder before milking, and positive milk color and consistency on the PL test (California Mastitis Test variant method) (PL Tester, Nihon Zenyaku Kogyo K.K., Koriyama, Japan). The study period was from June to December 2022. The 42 cows were randomly assigned to the MX-treated group (T group n=21) or the control group (C group n=21). The body condition score (BCS) of the T group averaged 2.76 ± 0.37 standard deviation, while that of the C group was 2.91 ± 0.24. The mean parity for the T group was 2.4 ± 1.6 standard deviation, while that for the C group was 2.2 ± 1.1. Days in milk (DIM) in the T group averaged 238.8 days (2–727 days, median 250.9 days) and that in the C group was 338.4 days (17–581 days, median 305 days), with no significant difference (P=0.0761) between the two groups. The rectal temperature and examination of each cow were conducted on days 0–3 (the first to the fourth sick day). Treatment of the affected quarter in both groups was administered once a day for 3 days using cephazolin ointment (cephazolin 150 mg/3 g, Cefazolin L “Fujita,” Fujita Pharmaceuticals, Tokyo, Japan).
Administration of analgesics
On day 0, the T group received 0.5 mg/kg subcutaneous (SC) injection of MX (Metacam 2% injection solution; Boehringer Ingelheim Animal Health Japan Co., Ltd., Tokyo, Japan) whereas the C group received a 15 mL SC injection of a placebo (saline solution, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).
Udder pain measurements
The measurement of pain in the affected quarter was done by applying pressure to the quarter with a measuring meter to indicate pain. Tenderness in the affected udder (UT) was measured using a digital concentrator high-precision pull manometer (SHAHE Co., Zhejiang, China). Pressure gauges were pressed against five locations on the quarter, and the average pressure applied by kicking, weight shifting, rear foot stomping, and other movements indicative of hind limb discomfort were indicated as UT in kg. Measurements were performed daily from day 0 to day 3. In addition, the UT of seven healthy cohabiting cow quarters (LS: 0.80 ± 0.20) was measured in a preliminary study.
Milk sampling
Milk from the affected quarters was collected daily from day 0 to day 3. The somatic cell count (SCC) was measured immediately after milk collection using the simple somatic cell analyzer ADAM SCC, (NanoEnTek Inc.Co., Seoul, Korea) and expressed as a linear score (LS)(LS=log2 (somatic cell count/100) +3). A 100 µL sample of day 0 milk was applied to a 5% sheep blood agar medium (Trypcase soy 5% sheep blood agar medium (TSS), Sysmex Biomérieux Co., Ltd., Tokyo, Japan) and incubated at 37°C for 24–48 hr. Bacterial species were identified either by 16SrDNA sequence analysis or mass spectrometry, and partial 16S rDNA sequences (approximately 0.8 kbp) were determined using the Bacterial 16S rDNA PCR Kit Fast (800; Takara Bio Inc., Tokyo, Japan). The sequences were analyzed based on their homogeneity using the Basic Local Alignment Search Tool (BLAST) program. AXIMA confidence (Shimadzu, Co., Kyoto, Japan) was used to identify bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Data analysis was performed by Saramis software. Whey was centrifuged (4°C, RCF 3,426 × g, 10 min) to remove milk fat and stored at −80°C until measurement.
Determination of the whey PGE2 concentrations
Whey PGE2 concentrations were measured using a commercially available ELISA kit (Prostaglandin E2 ELISA kit monoclonal, Inter-assay 6.6%CV, assay sensitivity15 pg/mL; Cayman Co., Ltd., Ann Arbor, MI, USA).
Determination of the whey BK concentrations
Whey BK levels were measured using a commercially available ELISA kit (Bradykinin ELISA kit, Inter-assay 6.8%CV, assay sensitivity 24.8 pg/mL; Enzo Life Sciences, Inc., Farmingdale, NY, USA).
Measurement of the MY before disease onset and after healing
MY prior to disease onset was determined using herd test data and the sum of the farmer’s morning and evening milk-meters. The cure was determined when there was no recurrence of mastitis after 2 weeks from the last day of mastitis ointment injection, no symptoms in the affected quarter, and a negative PL test for milk. Post-healing MY was measured by summing morning and evening milk-meters at least two weeks after treatment.
Statistical analysis
EZR (Ver. 1.61, Jichi Medical University, Saitama, Japan) was used for statistical analysis. A student t-test was used to determine significant differences in the number of days after healing between the two groups. The χ2 test was used to verify the independence of the T and C groups by cause species. Statistics on MY before the onset of illness and after healing and rectal temperatures on day 0 for both groups and the DIM of both groups were obtained using the Mann-Whitney U test. A two-way analysis of variance was used to compare UT, LS, whey PGE2 concentrations and whey BK concentrations and the Tukey-Kramar test was used for the post-hoc test. Correlations between test items were assessed using Pearson’s cumulative correlation coefficient. A P-value <0.05 All data was considered as statistically significant.
RESULTS
Clinical findings in the T and C groups
All cows in both groups had appetites and vigor. The rectal temperature on day 0 of the T group (38.9 ± 0.2°C) was not significantly different (P=0.7290) from that of the C group (38.9 ± 0.1°C). All the cows in both groups experienced fever, swelling, pain, induration in the affected quarters, and a positive PL test for milk.
ACM-causing bacteria
The causative species of ACM in the T and C groups are listed in Table 1. There was no significant (P=0.0572) independence between the two groups.
Table 1. Bacteria causing mastitis in the meloxicam-treated (T group) and control (C group) groups.
| Pathogen | T group | C group |
|---|---|---|
| Coliform | 10 | 2 |
| Streptococcus sp. | 4 | 5 |
| CNS | 4 | 4 |
| Gram-positive bacillus | 2 | 7 |
| SA | 1 | 0 |
| Oxidase-positive gram-negative bacillus | 0 | 2 |
| Aerococcus | 0 | 1 |
| Total | 21 | 21 |
CNS, coagulase negative Staphylococci; SA, Staphylococcus aureus.
Change in MY before disease onset and after healing
The mean time to cure in the T group was 16.8 days ± standard deviation 1.9 days, and that in the C group was 17.0 ± 3.4 days, which was not significant between the two groups (P=0.4126). The measurement of milk yield after healing was similar between the two groups, with healing confirmed before the evening milking and the total milk yield in the morning and evening of that day. The T group showed little change in MY before disease onset or compared to after healing. In contrast, the MY in the C group were significantly (P<0.01) reduced by approximately 6 kg after healing (Fig. 1).
Fig. 1.
Comparison of the milk yield between the meloxicam-treated group (T group) and control group (C group) before the onset of disease and after healing. Error line: Mean ± SEM. **: P<0.01.
Changes in UT in the affected quarter
The UT of the healthy quarter section from the preliminary study was 2.87 ± 0.15 kg. The UT showed reduced udder tenderness from day 0 to day 3 in both groups (Fig. 2). Pain in the T group was more alleviated than that in the C group, as UT on day 3 in the T group (2.33 ± 0.08 kg) was significantly (P<0.01) higher than that in the C group (1.75 ± 0.14 kg; Fig. 2).
Fig. 2.
Change in tenderness of the affected quarter in acute clinical mastitis in the meloxicam-treated group (T group) and the control group (C group). Error line: Mean ± SEM. T group: A significant increase in pressure (kg) was observed on days 1, 2, and 3 respectively, compared with that on day 0. C group: A significant increase in pressure (kg) was observed on days 2 and 3 compared with that on day 0. Significant difference between the two groups. **: P<0.01.
Change in milk LS
The LS in milk decreased significantly (P<0.01) on day 3 compared to day 0 in the T group (Fig. 3). Additionally, a significant decrease (P<0.01) on day 3 versus day 1 was identified in the C group (Fig. 3). There was no significant difference (P=0.2823) between the two groups.
Fig. 3.
Change in the linear score of affected quarter in acute clinical mastitis in the meloxicam-treated group (T group) and control group (C group). Error line: Mean ± SEM. The linear score in milk decreased significantly (P<0.01) on day 3 vs. day 0 in the T group. There was also a significant (P<0.01) decrease on day 3 vs. day 1 in the C group.
Changes in whey PGE2 concentrations
The whey PGE2 concentration decreased significantly (P<0.01) from day 0 to day 3 in both groups (Fig. 4A). The PGE2 concentration on day 0 was set at 1, and the reduction rate on day 1 was 35.4% for the T group and 38.4% for the C group. On day 2, the reduction rate for the T group (27.9%) was lower than that for the C group (35.7%) (Fig. 4B). However, there was no significant difference between the two groups.
Fig. 4.
Changes in the whey prostaglandin E2 (PGE2) concentration of the affected quarters in acute clinical mastitis from the onset of acute mastitis (day 0) to day 3 in the meloxicam-treated group (T group) and control group (C group). (A) Shows the change in measured PGE2 concentration. (B) Shows the percentage change in PGE2 concentration from day 1 to day 3 with day0 PGE2 concentration as 1. Error line: Mean ± SEM. (A) The whey prostaglandin E2 concentration in whey significantly (P<0.01) from day 0 to day 3 in both groups. (B) The PGE2 concentration on day 0 was set at 1, and the reduction rate on day 1 was 35.4% for T group and 38.4% for C group. On day 2, the reduction rate for T group (27.9%) was lower than that for C group (35.7%). However, there was no significant difference between the two groups.
Changes in whey BK concentrations
The BK concentration in the whey of the T group ranged from 3.0 to 4.7 pg/mL from Day 0 to Day 2 and reached 12.2 pg/mL on Day 3. The BK concentration in the whey of the C group ranged from 1.9 to 4.7 pg/mL from day 0 to day 3. The whey BK concentrations was significantly higher on day 3 in the T group, compared to day 0 to day 2, and was significantly higher than that in the C group (Fig. 5).
Fig. 5.
Changes in the whey bradykinin concentration of the affected quarters in acute clinical mastitis from the onset of acute clinical mastitis (day 0) to day 3 in the meloxicam-treated group (T group) and control group (C group). Error line: Mean ± SEM. The change in the whey BK concentration was significantly higher on day 3 in the T group than from day 0 to day 2, and was significantly higher than that in the C group. Significant difference between the two groups *: P<0.05.
Correlation of UT, LS, and whey PGE2 and BK concentrations at each illness date
Table 2 shows the correlation between UT, LS, and whey PGE2 and BK concentrations on each disease day for both groups. On day 0, a significant (P<0.05) positive correlation was found between whey PGE2 concentration and UT in the T group. A significant negative correlation was found between whey PGE2 concentration and LS on day 1 in the T group. A significant (P<0.05) positive correlation was also observed between whey PGE2 and whey BK concentrations and LS on day 3 in the T group. A significant positive correlation (PGE2 vs. LS: P<0.05, BK vs. LS: P<0.01) between whey PGE2 levels and whey BK concentrations and LS on day 2 in the C group. Moreover, a significant positive correlation (P<0.05) between whey PGE2 concentration and BK concentration was identified. Finally, a significant positive correlation (P<0.05) was observed between whey PGE2 concentration and LS on day 3 in the T and C groups.
Table 2. Correlation between udder tenderness, linear score, prostaglandin E2 concentration in whey and bradykinin concentration in whey of meloxicam treated group (T group) and control group (C group) on each day of illness.
DISCUSSION
McDougall et al. reported that the bacteriological cure rate of affected breasts was better when MX was administered for the treatment of moderate to mild clinical mastitis that developed within 120 days postpartum compared to the control group [9]. In our study, there was no decrease in MY after healing in the T group compared to that before disease onset. In contrast, a significant decrease in milk production was identified in the C group after healing compared to that before the onset of the disease. The administration of MX at the initial presentation of ACM is thought to have promoted anti-inflammatory and analgesic effects in the mammary gland, thus sparing the patients from decreased milk production after healing.
Upon experimental Escherichia coli (E. coli) injection into the udder of dairy cows, udder touch during the acute 12 to 24 hr period can generate pain responses such as kicking, flinching, stepping, and weight shifting [13]. Ginger et al. reported that the intramuscular injection of the NSAID ketoprofen, into dairy cows that had received an intramammary injection with LPS significantly reduced pain responses (i.e., hoof raising and kicking with hind legs) [6]. Fitzpatrick et al. measured UT in cows experimentally injected with intramammary LPS and reported that those treated with MX presented no change in pain, whereas cows that received placebo showed significant pain after 6 hr [5]. The present study showed by stimulation with a tenderness meter that a significant reduction in pain (P<0.01) occurred in the T group on day 3, compared with that in the C group. This suggests that the administration of MX at the beginning of ACM improved the pain of the affected quarter on day 3.
Caldeira et al. compared the changes in milk SCC in cows that were experimentally injected with intramammarily LPS and intravenously injected with MX to those injected with intramammarily LPS alone. Their results showed that MX did not significantly decrease SCC, with similar values 9 hr after injection [3]. They also reported that injecting MX alone into the udder did not affect SCC [3]. In the present study, the LS for both groups were significantly lower (P<0.01) on days 0 and 1 compared to day 3. The subcutaneous injection of MX in LPS-induced clinical mastitis in Fitzpatrick et al. [5] showed temporal changes in milk SCC, similar to those found in the present study. Using udder biopsy, Caldeira et al. showed that the relative dependence of TNF-α and IL-1 mRNA on LPS stimulation in the mammary gland was not significantly different with or without MX administration [3]. The lack of a significant difference in LS changes between the two groups in the current study suggests that MX had no direct effect on milk SCCs, as it blocked COX-2 downstream in the inflammatory process.
Caldeira et al. reported that MX inhibits PGE2 synthesis upon bovine mammary gland cell stimulation with LPS from E. coli or lipoteichoic acid from Staphylococcus aureus (S. aureus) [2]. In our study, the PGE2 levels decreased over time in the T and C groups and did not differ significantly between the groups. The ratio of day 1 and day 2 to day0 PGE2 concentrations was lower in the T group than in the C group, although the difference was not significant. Pezeshki et al. reported that when dairy cows were experimentally infected with E. coli in the udder after calving, udder PGE2 levels were associated with bacterial growth and intensity of the inflammatory response, which in turn were associated with the severity of systemic clinical signs [11]. This suggests that no difference in the PGE2 levels between the two groups occurred in this study because the ACM presented solely with localized symptoms in the affected quarter, without systemic symptoms. The fact that the ratio of day 1 and day 2 PGE2 concentrations to day 0 was lower in the T group than in the C group was presumably indicative of a drug effect of MX.
BK is a degradation product of high-molecular-weight kininogen in the kinin cascade catalyzed by kallikrein, which increases vasodilation, vascular permeability, and pain during tissue inflammation [12]. In our study, the whey BK concentrations of the T group ranged from 3.0–4.7 pg/mL from day 0 to day 2 and reached 12.2 pg/mL on day 3. The whey BK concentrations of the C group ranged from 1.9–4.7 pg/mL from day 0 to day 3. Eshraghi et al. reported that milk BK levels were 800 pg/mL in cases of clinical bovine mastitis, with severe and discolored milk coagulation, unmeasurable SCC, swelling, pain, edema, and induration [4]. This suggests that the patients in the present study presented milder symptoms than those of Eshraghi et al. Therefore, no significant increase in BK levels were identified in either group.
S. aureus has been reported to increase secretion of cytokines in bovine neutrophils. In dairy cow infected with wild-type S. aureus (SA113) and a lipoprotein-deficient strain of S. aureus (Δlgt), Δlgt was less effective than SA113 in inducing the production of IL-1β, IL-6, IL-8, IL-10, and PGE2 within neutrophils [14]. In our study, a significant negative correlation (P<0.05) was found between whey PGE2 concentrations and LS on day 1 in the T group but not in the C group. This was thought to indicate that MX administration suppressed PGE2 production from mammary tissue and negatively correlated with the number of neutrophils that migrated from within the blood vessels due to inflammation. Additionally, a significant positive correlation (P<0.05) was observed between the whey PGE2 concentration and LS on day 2 in the C group but not in the T group. The anti-inflammatory and antipyretic properties of isopropyl vanillate (ISP-VT) in mice have been reported; the main mechanism of ISP-VT-induced anti-inflammatory activity is the inhibition of COX-2, which reduces foot edema induced by BK, histamine, and PGE2, and decreases neutrophil recruitment [10]. In our study, there was a significant positive correlation between whey PGE2 concentration and LS on day 2 in group C but not in group T. These findings suggest that COX-2 inhibition in mammary tissue by MX suppresses PGE2 synthesis and decreases neutrophil mobilization. This suggests that the number of neutrophils migrating from within the blood vessels increased in group C due to the inflammatory response of the mammary tissue and that PGE2 production was enhanced by inflammation-induced mammary tissue damage. In both groups, a positive correlation was observed between whey PGE2 concentration and LS on day 3. This is presumably due to the continued inflammatory response from day 2 in the C group. In addition, in the T group, it is presumed that this is because the pharmacological effect time of MX (28.8 hr after administration of the effect time) has elapsed.
The cost of MX in the T group was about 3,765 yen for only one dose. Six kg of milk loss after healing in the C group was 120 yen/kg, which was calculated to be 720 yen per day and a loss of 12,240 yen per day until healing (17 days), suggesting that the T group was cost-effective compared to the C group from an economic standpoint.
In conclusion, administration of MX in ACM that showed symptoms only in the udder without systemic symptoms resulted in better recovery of MY after healing and reduced bovine udder pain. Administering MX to ACM without systemic symptoms has economic benefits and can improve animal welfare.
CONFLICT OF INTEREST
This study was funded by Boehringer Ingelheim and Animal Health Japan Co., Ltd.
Acknowledgments
The authors thank Boehringer Ingelheim and Animal Health Japan Co., Ltd. for their assistance with this study.
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