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Animal Science Journal (2015) 86, 153–158
doi: 10.1111/asj.12269
ORIGINAL ARTICLE Dynamics of lingual antimicrobial peptide, lactoferrin concentrations and lactoperoxidase activity in the milk of cows treated for clinical mastitis Kazuhiro KAWAI,1 Kiyoshi KOREMATSU,2 Kiyoshi AKIYAMA,3 Miki OKITA,4 Yukinori YOSHIMURA4 and Naoki ISOBE4 1
School of Veterinary Medicine, Azabu University, Sagamihara, 2Kirishima Veterinarians, Kobayashi, 3Kanagawa Prefectural Livestock Industry Technology Center, Hiratsuka and 4Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan
ABSTRACT The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day −6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days −6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 ( 5 × 106 cells/mL and low-SCC group: < 5 × 106 cells/mL). Moreover, two types of changes were observed in SCC in cows treated with antibiotics: a decrease (< 5 × 105 cells/mL) in SCC was observed in cows with the first type after the treatment (low-SCC group), while SCC in cows with the second type remained high (> 5 × 105 cells/mL: high-SCC group). Therefore, cows were divided into these two types to examine changes in innate immune factors in more detail.
Examination of milk samples Milk samples were divided into two aliquots; the first aliquot was spread on a glass slide to measure SCC following the Breed method (Schalm et al. 1971). The second aliquot of © 2014 Japanese Society of Animal Science
milk was defatted by centrifugation at 1700 × g for 30 min at 4°C and stored at −30°C until the measurement of LAP and LF concentrations and LPO activity (Isobe et al. 2013). The enzyme immunoassay used to determine LAP was performed as previously described (Isobe et al. 2009b). A 96-well microtiter plate was coated with 2 μg/mL of an antiLAP antibody (Isobe et al. 2009c). Ten-fold diluted skim milk was cultured with horseradish peroxidase-labeled LAP (Isobe et al. 2009c) for 3 h at room temperature. After the incubation with tetramethyl benzidine (TMB), optical density was measured at a wavelength of 655 nm. The sensitivity and recovery of LAP were 0.05 ng/mL and 100–115%, respectively. The coefficients of variation for intra- and inter-assay variations were 6.5% and 13.5%, respectively. LF concentrations were measured by an ELISA quantification set following a commercial protocol (Bethyl Laboratories, Inc., Montgomery, TX, USA). Milk LPO activity was determined as described by Isobe et al. (2011). Defatted milk was mixed with TMB solution and incubated at 37°C for 30 min. After a brief centrifugation at 6000 × g for 1 min, the optical density of the supernatant was measured at 655 nm. Lactoperoxidase (Sigma, St. Louis, MO, USA) at a concentration of 0–10 U was used as the standard (one unit formed 1 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 20°C). Milk samples were cultured on 5% sheep blood agar and grown bacteria were identified according to the procedure described by the National Mastitis Council (Hogan et al. 1999).
Statistical analysis Differences in LAP and LF concentrations and LPO activity between groups were analyzed by Student’s t-test. A probability of < 0.05 was considered significant.
RESULTS Three cows in the antibiotics group and two cows in the control group were infected with Staphylococcus aureus. Streptococcus dysgalactiae was observed in three cows in the antibiotics group. When cows with and without infection with Staphylococcus aureus or Streptococcus dysgalactiae were divided, no significant difference was observed in LAP and LF concentrations, LPO activity or SCC. The innate immune factors in milk treated with antibiotics were compared with those in milk not treated, and the results are shown in Figure 1. The concentration of LAP and LPO activity did not change during the experimental period (Fig. 1a,b). The concentration of LF in non-treated cows also did not change, but markedly decreased in treated cows after day 8 (Fig. 1c). Significant differences (P < 0.05) were observed in LF concentrations between groups on days 10 and 12. SCC also decreased after the antibiotic treatment, and SCC on days 6 to 12 was significantly different (P < 0.05) from that in non-treated cows (Fig. 1d). To examine whether the severity of mastitis at the onset of the experiment is related to innate immune factors, cows treated with antibiotics were divided into Animal Science Journal (2015) 86, 153–158
THERAPY AND LAP, LF IN MASTITIC MILK
20
30
LPO activity (U/mL)
LAP concentration (ng/mL)
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10
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Figure 1 Changes in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations, lactoperoxidase (LPO) activity and somatic cell count (SCC) in the milk of cows treated with antibiotics and those not treated. Circles and squares mean cows treated with antibiotics and not treated, respectively. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
two groups based on SCC on the day of the treatment (high-SCC group: > 5 × 106 cells/mL and low-SCC group: < 5 × 106 cells/mL). SCC in cows in the lowSCC group decreased until day 4, and no further decrease was observed thereafter (Fig. 2d). However, SCC continued to decrease in cows in the high-SCC group until day 12. Significant differences (P < 0.05) were observed in SCC between groups on days 2–8, but not on days 10–12. High LAP concentrations were maintained in the low-SCC group during the whole experimental period and those on days 0, 2 and 6 were significantly higher (P < 0.05) than those in the highSCC group (Fig. 2a). However, LPO activity and LF concentrations were higher in the high-SCC group on day 0 than in the other group during the whole experimental period (Fig. 2b,c). LPO activity on days 0 and 12 were significantly higher (P < 0.05) in cows from the high-SCC group than in those of the low-SCC group (Fig. 2b). To examine whether recovery output after antibiotics treatment is related to innate immune factors, Animal Science Journal (2015) 86, 153–158
treated cows were further divided into two groups based on SCC after the treatment (low-SCC group: SCC decreased (< 5 × 105 cells/mL) after the treatment and high-SCC group: SCC remained high (> 5 × 105 cells/mL)). LAP concentrations in cows from the high-SCC group tended slightly higher on days 10 and 12 (Fig. 3a). In contrast, LPO activity in cows of the high-SCC group was lower than that in cows from the low-SCC group during the whole experimental period, and this difference was significant (P < 0.05) on day 10 (Fig. 3b). LF concentrations were higher (P < 0.05) in cows from the high-SCC group than in those of the low SCC group on day 12 (Fig. 3c).
DISCUSSION The antibiotic treatment reduced SCC on days 6–12, which suggested that antibiotics can decrease SCC, leading to recovery from mastitic inflammation in mammary glands. Similarly, the concentrations of © 2014 Japanese Society of Animal Science
156 K. KAWAI et al.
(a)
25 *
*
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10 > 5 × 106 cells/ml SCC < 5 × 106 cells/ml SCC
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-6 -4 -2 0 2 4 6 8 10 12 Day (d)
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*
* *
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Figure 2 Differences in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity in milk between cows with < 5 × 106 or > 5 × 106 cells/mL of somatic cell count (SCC) on day 0. Circles and squares mean cows with SCC of > 5 × 106 and < 5 × 106 cells/mL on day 0, respectively. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
LF were significantly lower in the antibiotics group than in the control group. However, the concentration of LAP and LPO activity were maintained after the antibiotic treatment. These results suggest that the secretion of LF may be associated with high SCC, but not LAP concentrations or LPO activity. LF is secreted from epithelial cells (Huang et al. 2012) or leukocytes, although in this case, LF may have mainly come from leukocytes; therefore, LF concentrations decreased concomitantly with the decrease in SCC. To examine whether recovery output after antibiotics treatment is related to innate immune factors, cows in the antibiotics group were further classified into a group in which SCC was decreased < 5 × 105 cells/mL after the treatment and a group in which SCC was not decreased. LF concentrations were significantly higher in the > 5 × 105 cells/mL group than in the < 5 × 105 cells/mL group on day 12 only. These results further support the above-described suggestion that © 2014 Japanese Society of Animal Science
the secretion of LF is associated with high SCC. In contrast, LPO activity was significantly higher in the < 5 × 105 cells/mL group on day 10 than in the > 5 × 105 cells/mL group. This result indicates that less inflammation led to activity of LPO being high in the milk of dairy cows. LPO was previously shown to be produced by leukocytes and mammary epithelial cells (Isobe et al. 2011). Therefore, the ratio of leukocytes to the total number of SCC may differ between groups; the low SCC group may contain a higher number of leukocytes, leading to high LPO activity. Changes in the ratio of cell types in SCC has also been previously reported (Rainard et al. 2013). The severity of mastitis at the onset of the experiment may be related to innate immune factors after the antibiotic treatment. Therefore, cows treated with antibiotics were further classified as having higher SCC (> 5 × 106 cells/mL) or lower SCC (< 5 × 106 cells/ mL) on day 0. SCC was significantly higher on days 2–8 in cows with higher SCC on day 0 than that in Animal Science Journal (2015) 86, 153–158
THERAPY AND LAP, LF IN MASTITIC MILK
25
40 30 20 < 5 × 105 cells/ml SCC
10
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LAP concentration (ng/mL)
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Figure 3 Differences in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity in milk between cows with and without recovered somatic cell count (SCC) (< 5 × 105 cells/mL) after the antibiotic treatment. Circles mean cows in which SCC decreased to < 5 × 105 cells/mL after the antibiotic treatment. Squares mean cows in which SCC remained > 5 × 105 cells/mL after the antibiotic treatment. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
cows with lower SCC. Therefore, mastitis in cows with high SCC may take a longer time to recover. LPO activity on days 0 and 12 were significantly higher in cows with higher SCC on day 0 than in those with lower SCC. Higher SCC containing leukocytes may lead to the greater secretion of LPO in milk. In contrast, LAP concentrations were significantly lower in cows with higher SCC on day 0 than in those with lower SCC. Since LAP is secreted from the epithelium into milk (Isobe et al. 2009a,c), mammary glands may have been damaged in the > 5 × 106 cells/mL group, whereas those in the < 5 × 106 cell/mL group had more intact epithelial cells, which produced high concentrations of LAP. Severe mastitis has been shown to induce the death of the alveolar epithelium in mammary glands (Kitchen et al. 1980). In conclusion, the present results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activAnimal Science Journal (2015) 86, 153–158
ity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factors in milk of cows treated for clinical mastitis.
ACKNOWLEDGMENT This work was supported by JSPS Grants-in-Aid for Scientific Research to N. Isobe (24580410).
REFERENCES Arnold RR, Cole MF, McGhee JR. 1977. A bactericidal effect for human lactoferrin. Science 197, 263–265. Bosch EH, van Doorne H, de Vries S. 2000. The lactoperoxidase system: the influence of iodide and the chemical and antimicrobial stability over the period of about 18 months. Journal of Applied Microbiology 89, 215– 224. Ganz T. 2003. Defensins: antimicrobial peptides of innate immunity. Nature Review Immunology 3, 710–720. © 2014 Japanese Society of Animal Science
158 K. KAWAI et al.
Hogan JS, González RN, Harmon RJ, Nickerson SC, Oliver SP, Pankey JW, Smith KL. 1999. Laboratory Handbook on Bovine Mastitis, revised edn. National Mastitis Council Inc, Madison, WI. Huang YQ, Morimoto K, Hosoda K, Yoshimura Y, Isobe N. 2012. Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows. Veterinary Immunology and Immunopathology 145, 499–504. Hurley WL, Rejman JJ. 1993. Bovine lactoferrin in involuting mammary tissue. Cell Biology International 17, 283– 289. Isobe N, Hosoda K, Yoshimura Y. 2009c. Immunolocalization of lingual antimicrobial peptide (LAP) in the bovine mammary gland. Animal Science Journal 80, 446–450. Isobe N, Kubota H, Yamasaki A, Yoshimura Y. 2011. Lactoperoxidase activity in milk is correlated to somatic cell count in dairy cows. Journal of Dairy Science 94, 3868– 3874. Isobe N, Morimoto K, Nakamura J, Yamasaki A, Yoshimura Y. 2009b. Intramammary challenge of lipopolysaccharide stimulates secretion of lingual antimicrobial peptide into milk of dairy cows. Journal of Dairy Science 92, 6046–6051. Isobe N, Nakamura J, Nakano H, Yoshimura Y. 2009a. Existence of functional lingual antimicrobial peptide (LAP) in bovine milk. Journal of Dairy Science 92, 2691–2695. Isobe N, Shibata A, Kubota H, Yoshimura Y. 2013. Lingual antimicrobial peptide and lactoferrin concentrations and lactoperoxidase activity in bovine colostrum are associated with subsequent somatic cell count. Animal Science Journal 84, 751–756. Kawai K, Akamatsu H, Obayashi T, Nagahata H, Higuchi H, Iwano H, et al. 2013. Relationship between concentration
© 2014 Japanese Society of Animal Science
of lingual antimicrobial peptide and somatic cell count in milk of dairy cows. Veterinary Immunology and Immunopathology 153, 298–301. Kitchen BJ, Middleton G, Durward IG, Andrews RJ, Salmon MC. 1980. Mastitis diagnostic tests to estimate mammary gland epithelial cell damage. Journal of Dairy Science 63, 978–983. National Research Council. 2001. Nutrient Requirements of Dairy Cattle, 7th revised edn. National Academic Press, Washington, DC. Rainard P, Cunha P, Bougarn S, Fromageau A, Rossignol C, Gilbert FB, Berthon P. 2013. T helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland. PLoS ONE 8, e63471. Schalm OW, Carroll EJ, Jain NC. 1971. Number and types of somatic cells in normal and mastitic milk. In: Schalm OW, Carroll EJ, Jain NC (eds), Bovine Mastitis, 1st edn. pp. 94–127. Lea and Febiger, Philadelphia, PA. Schonwetter BS, Stolzenberg ED, Zasloff MA. 1995. Epithelial antibiotics induced at sites of inflammation. Science 267, 1645–1648. Shin K, Hayasawa H, Lönnerdal B. 2001. Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase hydrogen peroxide-thiocyanate antimicrobial system. Journal of Applied Microbiology 90, 489–493. Swanson K, Gorodetsky S, Good L, Davis S, Musgrave D, Stelwagen K, et al. 2004. Expression of a β-defensin mRNA, lingual antimicrobial peptide, in bovine mammary epithelial tissue is induced by mastitis. Infection and Immunnity 72, 7311–7314. Weinberg ED. 1978. Iron and infection. Microbiology Review 42, 45–66.
Animal Science Journal (2015) 86, 153–158
Animal Science Journal (2015) 86, 153–158
doi: 10.1111/asj.12269
ORIGINAL ARTICLE Dynamics of lingual antimicrobial peptide, lactoferrin concentrations and lactoperoxidase activity in the milk of cows treated for clinical mastitis Kazuhiro KAWAI,1 Kiyoshi KOREMATSU,2 Kiyoshi AKIYAMA,3 Miki OKITA,4 Yukinori YOSHIMURA4 and Naoki ISOBE4 1
School of Veterinary Medicine, Azabu University, Sagamihara, 2Kirishima Veterinarians, Kobayashi, 3Kanagawa Prefectural Livestock Industry Technology Center, Hiratsuka and 4Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan
ABSTRACT The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day −6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days −6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 ( 5 × 106 cells/mL and low-SCC group: < 5 × 106 cells/mL). Moreover, two types of changes were observed in SCC in cows treated with antibiotics: a decrease (< 5 × 105 cells/mL) in SCC was observed in cows with the first type after the treatment (low-SCC group), while SCC in cows with the second type remained high (> 5 × 105 cells/mL: high-SCC group). Therefore, cows were divided into these two types to examine changes in innate immune factors in more detail.
Examination of milk samples Milk samples were divided into two aliquots; the first aliquot was spread on a glass slide to measure SCC following the Breed method (Schalm et al. 1971). The second aliquot of © 2014 Japanese Society of Animal Science
milk was defatted by centrifugation at 1700 × g for 30 min at 4°C and stored at −30°C until the measurement of LAP and LF concentrations and LPO activity (Isobe et al. 2013). The enzyme immunoassay used to determine LAP was performed as previously described (Isobe et al. 2009b). A 96-well microtiter plate was coated with 2 μg/mL of an antiLAP antibody (Isobe et al. 2009c). Ten-fold diluted skim milk was cultured with horseradish peroxidase-labeled LAP (Isobe et al. 2009c) for 3 h at room temperature. After the incubation with tetramethyl benzidine (TMB), optical density was measured at a wavelength of 655 nm. The sensitivity and recovery of LAP were 0.05 ng/mL and 100–115%, respectively. The coefficients of variation for intra- and inter-assay variations were 6.5% and 13.5%, respectively. LF concentrations were measured by an ELISA quantification set following a commercial protocol (Bethyl Laboratories, Inc., Montgomery, TX, USA). Milk LPO activity was determined as described by Isobe et al. (2011). Defatted milk was mixed with TMB solution and incubated at 37°C for 30 min. After a brief centrifugation at 6000 × g for 1 min, the optical density of the supernatant was measured at 655 nm. Lactoperoxidase (Sigma, St. Louis, MO, USA) at a concentration of 0–10 U was used as the standard (one unit formed 1 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 20°C). Milk samples were cultured on 5% sheep blood agar and grown bacteria were identified according to the procedure described by the National Mastitis Council (Hogan et al. 1999).
Statistical analysis Differences in LAP and LF concentrations and LPO activity between groups were analyzed by Student’s t-test. A probability of < 0.05 was considered significant.
RESULTS Three cows in the antibiotics group and two cows in the control group were infected with Staphylococcus aureus. Streptococcus dysgalactiae was observed in three cows in the antibiotics group. When cows with and without infection with Staphylococcus aureus or Streptococcus dysgalactiae were divided, no significant difference was observed in LAP and LF concentrations, LPO activity or SCC. The innate immune factors in milk treated with antibiotics were compared with those in milk not treated, and the results are shown in Figure 1. The concentration of LAP and LPO activity did not change during the experimental period (Fig. 1a,b). The concentration of LF in non-treated cows also did not change, but markedly decreased in treated cows after day 8 (Fig. 1c). Significant differences (P < 0.05) were observed in LF concentrations between groups on days 10 and 12. SCC also decreased after the antibiotic treatment, and SCC on days 6 to 12 was significantly different (P < 0.05) from that in non-treated cows (Fig. 1d). To examine whether the severity of mastitis at the onset of the experiment is related to innate immune factors, cows treated with antibiotics were divided into Animal Science Journal (2015) 86, 153–158
THERAPY AND LAP, LF IN MASTITIC MILK
20
30
LPO activity (U/mL)
LAP concentration (ng/mL)
(a)
20
10
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(b)
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*
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Figure 1 Changes in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations, lactoperoxidase (LPO) activity and somatic cell count (SCC) in the milk of cows treated with antibiotics and those not treated. Circles and squares mean cows treated with antibiotics and not treated, respectively. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
two groups based on SCC on the day of the treatment (high-SCC group: > 5 × 106 cells/mL and low-SCC group: < 5 × 106 cells/mL). SCC in cows in the lowSCC group decreased until day 4, and no further decrease was observed thereafter (Fig. 2d). However, SCC continued to decrease in cows in the high-SCC group until day 12. Significant differences (P < 0.05) were observed in SCC between groups on days 2–8, but not on days 10–12. High LAP concentrations were maintained in the low-SCC group during the whole experimental period and those on days 0, 2 and 6 were significantly higher (P < 0.05) than those in the highSCC group (Fig. 2a). However, LPO activity and LF concentrations were higher in the high-SCC group on day 0 than in the other group during the whole experimental period (Fig. 2b,c). LPO activity on days 0 and 12 were significantly higher (P < 0.05) in cows from the high-SCC group than in those of the low-SCC group (Fig. 2b). To examine whether recovery output after antibiotics treatment is related to innate immune factors, Animal Science Journal (2015) 86, 153–158
treated cows were further divided into two groups based on SCC after the treatment (low-SCC group: SCC decreased (< 5 × 105 cells/mL) after the treatment and high-SCC group: SCC remained high (> 5 × 105 cells/mL)). LAP concentrations in cows from the high-SCC group tended slightly higher on days 10 and 12 (Fig. 3a). In contrast, LPO activity in cows of the high-SCC group was lower than that in cows from the low-SCC group during the whole experimental period, and this difference was significant (P < 0.05) on day 10 (Fig. 3b). LF concentrations were higher (P < 0.05) in cows from the high-SCC group than in those of the low SCC group on day 12 (Fig. 3c).
DISCUSSION The antibiotic treatment reduced SCC on days 6–12, which suggested that antibiotics can decrease SCC, leading to recovery from mastitic inflammation in mammary glands. Similarly, the concentrations of © 2014 Japanese Society of Animal Science
156 K. KAWAI et al.
(a)
25 *
*
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20
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0
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* *
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5
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4 6 Day
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Figure 2 Differences in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity in milk between cows with < 5 × 106 or > 5 × 106 cells/mL of somatic cell count (SCC) on day 0. Circles and squares mean cows with SCC of > 5 × 106 and < 5 × 106 cells/mL on day 0, respectively. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
LF were significantly lower in the antibiotics group than in the control group. However, the concentration of LAP and LPO activity were maintained after the antibiotic treatment. These results suggest that the secretion of LF may be associated with high SCC, but not LAP concentrations or LPO activity. LF is secreted from epithelial cells (Huang et al. 2012) or leukocytes, although in this case, LF may have mainly come from leukocytes; therefore, LF concentrations decreased concomitantly with the decrease in SCC. To examine whether recovery output after antibiotics treatment is related to innate immune factors, cows in the antibiotics group were further classified into a group in which SCC was decreased < 5 × 105 cells/mL after the treatment and a group in which SCC was not decreased. LF concentrations were significantly higher in the > 5 × 105 cells/mL group than in the < 5 × 105 cells/mL group on day 12 only. These results further support the above-described suggestion that © 2014 Japanese Society of Animal Science
the secretion of LF is associated with high SCC. In contrast, LPO activity was significantly higher in the < 5 × 105 cells/mL group on day 10 than in the > 5 × 105 cells/mL group. This result indicates that less inflammation led to activity of LPO being high in the milk of dairy cows. LPO was previously shown to be produced by leukocytes and mammary epithelial cells (Isobe et al. 2011). Therefore, the ratio of leukocytes to the total number of SCC may differ between groups; the low SCC group may contain a higher number of leukocytes, leading to high LPO activity. Changes in the ratio of cell types in SCC has also been previously reported (Rainard et al. 2013). The severity of mastitis at the onset of the experiment may be related to innate immune factors after the antibiotic treatment. Therefore, cows treated with antibiotics were further classified as having higher SCC (> 5 × 106 cells/mL) or lower SCC (< 5 × 106 cells/ mL) on day 0. SCC was significantly higher on days 2–8 in cows with higher SCC on day 0 than that in Animal Science Journal (2015) 86, 153–158
THERAPY AND LAP, LF IN MASTITIC MILK
25
40 30 20 < 5 × 105 cells/ml SCC
10
LPO activity (U/mL)
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Figure 3 Differences in lingual antimicrobial peptide (LAP), lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity in milk between cows with and without recovered somatic cell count (SCC) (< 5 × 105 cells/mL) after the antibiotic treatment. Circles mean cows in which SCC decreased to < 5 × 105 cells/mL after the antibiotic treatment. Squares mean cows in which SCC remained > 5 × 105 cells/mL after the antibiotic treatment. *Shows significant differences between the groups on the same day. Day 0 is the day on which the antibiotic treatment started.
cows with lower SCC. Therefore, mastitis in cows with high SCC may take a longer time to recover. LPO activity on days 0 and 12 were significantly higher in cows with higher SCC on day 0 than in those with lower SCC. Higher SCC containing leukocytes may lead to the greater secretion of LPO in milk. In contrast, LAP concentrations were significantly lower in cows with higher SCC on day 0 than in those with lower SCC. Since LAP is secreted from the epithelium into milk (Isobe et al. 2009a,c), mammary glands may have been damaged in the > 5 × 106 cells/mL group, whereas those in the < 5 × 106 cell/mL group had more intact epithelial cells, which produced high concentrations of LAP. Severe mastitis has been shown to induce the death of the alveolar epithelium in mammary glands (Kitchen et al. 1980). In conclusion, the present results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activAnimal Science Journal (2015) 86, 153–158
ity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factors in milk of cows treated for clinical mastitis.
ACKNOWLEDGMENT This work was supported by JSPS Grants-in-Aid for Scientific Research to N. Isobe (24580410).
REFERENCES Arnold RR, Cole MF, McGhee JR. 1977. A bactericidal effect for human lactoferrin. Science 197, 263–265. Bosch EH, van Doorne H, de Vries S. 2000. The lactoperoxidase system: the influence of iodide and the chemical and antimicrobial stability over the period of about 18 months. Journal of Applied Microbiology 89, 215– 224. Ganz T. 2003. Defensins: antimicrobial peptides of innate immunity. Nature Review Immunology 3, 710–720. © 2014 Japanese Society of Animal Science
158 K. KAWAI et al.
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