FULL PAPER Clinical Pathology Lactoferrin Concentrations in Milk from Normal and Subclinical Mastitic Cows Sei-ichi HAGIWARA 1), Kazuhiro KAWAI 2), Akira ANRI 3) and Hajime NAGAHATA 4) 1) Hokkaido-East Area Section, Hokkaido NOSAI, North 4 East 1, Chuo-ku, Sapporo, Hokkaido 060 0004, 2) Chubu Veterinary Clinical Center, Tokachi NOSAI, 35 12 Kisen Motoimatsu-cho, Obihiro, Hokkaido 080 2331, 3) Large Animal Clinic and Research Center, Hokkaido NOSAI, 612 Motonopporo, Ebetsu, Hokkaido 069 0805 and 4) Department of Animal Health, School of Veterinary Medicine, Rakuno Gakuen University, Bunkyodai-Midori 582, Ebetsu, Hokkaido 069 8501, Japan (Received 17 January 2001/Accepted 4 December 2002) ABSTRACT. The concentrations of lactoferrin (Lf) in quarter milk from normal lactating cows and subclinical mastitic cows were measured to determine whether the Lf concentration in milk is influenced by the age of the cow, the stage of lactation, number of milk somatic cells and the presence of pathogens. Lf concentrations in 111 quarter milk samples from 28 normal lactating cows and 270 quarte r milk samples from 198 subclinical mastitic cows were measured by means of a single radial immunodiffusion test. Lf concentrations (m eans ± standard deviations; logarithmic form) in normal cows and subclinical mastitic cows were 2.23 ± 0.39 and 2.70 ± 0.39, respectively. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows. T he mean milk Lf concentration (log) in normal lactating cows aged 5 years was lower than those in normal lactating cows aged 2 years (p<0.01) and 3 years (p<0.05). The results showed that the milk Lf concentration (log) is associated with age of the dairy cow (one-way analysis of variance test, p<0.01). The mean milk Lf concentration (log) in the latter lactational period tended to be higher than those in the peak and middle periods. Milk Lf concentrations (log) tended to be proportional to the level of the somatic cell count (SCC) score. Mean milk Lf concentrations (log) in subclinical mastitic cows infected with Staphylococcus aureus and with other streptococci species were significantly (p<0.01) higher than those in cows infected with coagulase-negative staphylococci and with Corynebacterium bovis. KEY WORDS: age, lactoferrin, mastitis, milk, pathogen. J. Vet. Med. Sci. 65(3): 319 323, 2003 Lactoferrin (Lf) is an iron-binding antimicrobial protein present in milk and on mucosal surfaces [18], and it is thought to play a role in preimmune host defense [3, 10]. Based on the strong association between the Lf concentration and occurrence of mastitis and based on the antibacterial property of Lf, it has been suggested that bovine milk Lf plays an important role in host defense against Escherichia coli (E. coli) mastitis [1, 5, 6, 14 17, 19, 20]. On the other hand, Lf is thought to have little antibacterial effect on Staphylococcus aureus (SA) or Streptococcus agalactiae (SAG) [14, 16]. There have been many reports on the antibacterial effects of Lf [4, 9, 11, 12, 14, 20]. Harmon et al. [7] reported that the milk Lf concentration is correlated with the somatic cell count, stage of lactation and amount of milk. But the normal concentration of Lf in cows, especially that in the period of lactation, has still not been clearly established. Previously reported normal milk Lf concentrations in the lactation period were 250 404 µg/ml [7, 22, 23]. The aim of this study was to determine normal milk Lf concentrations and to determine the factors affecting milk Lf, such as the age of the cow, stage of lactation, somatic cell count score and the bacterial species isolated from subclinical mastitic cows. MATERIALS AND METHODS Cows: A total of 2,937 quarter milk samples were collected from 741 normal lactating Holstein-Friesian dairy cows at 20 farms in Hokkaido, Japan. After milking, 5 to 10 ml of milk was collected from each quarter into 15-ml plastic tubes. The mean age of lactating cows used in this study was 3.5 years, and the mean daily milk production was 29.0 kg. Milk samples: The milk samples were classified on the basis of bacterial isolates, somatic cell count (SCC) score and lactational stage. The criteria for selection of normal milk samples were the absence of bacteria and an SCC of less than 71,000 cells/ml for a period of more than 2 months. The criteria for selection of milk samples from subclinical mastitic cows were the presence of only one kind of bacterial growth with over 1,000 colony forming units (CFU)/ml and no clinical findings of mastitis in the cow at the time of sampling. Isolated bacteria were identified according to the procedure recommended by the National Mastitis Council [13]. A total of 111 quarter milk samples were selected as normal milk samples, and the bacteria species isolated from subclinical mastitic cows were Staphylococcus aureus (SA) in 84 samples, Streptococcus agalactiae (SAG) in 8 samples, coagulase-negative staphylococci (CNS) in 92 samples, other streptococci (OS) in 61 samples, and Corynebacterium bovis (CB) in 25 samples. In addition, these samples were classified by the SCC score and the lactational stage. All of the samples were stored at 15 C until used for assays. Stage of lactation: The lactational stage was classified by the number of days from parturition as shown in Table 1. Somatic cell count (SCC) score: SCC in milk was determined with a Fossomatic cell counter [2] at the Hokkaido
320 S. HAGIWARA, K. KAWAI, A. ANRI AND H. NAGAHATA Official Approval Milking Cow Association, Sapporo, Japan. The SCC was converted into the SCC score by the number of somatic cells as shown in Tables 1 and 2. Measurement of milk Lf concentrations: Milk Lf concentrations were measured with a single radial immunodiffusion test kit (Bacteria Science Laboratory, Miyagi, Japan). Statistical analysis: The data on measured Lf concentrations (µg/ml) were transformed into logarithmic form. Distribution of the milk Lf concentration (log) was proved by chi-square test for goodness of fit. Homogeneities of variance were analyzed by Bartlett s test in groups classified by the age of the cows, stage of lactation, SCC score and bacterial species. According to the results of Bartlett s test, the differences in milk Lf concentrations (log) among groups were analyzed by one-way analysis of variance or the Kruskal-Wallis test. The milk Lf concentrations (log) among groups were analyzed by Bonferroni s test. The correlation coefficients among classified factors were analyzed by Speaman s correlation coefficient. A p value of less than 0.05 was considered significant. RESULTS Milk Lf concentrations in normal lactating cows and subclinical mastitic cows: Milk Lf concentrations (µg/ml) in normal lactating cows and subclinical mastitic cows ranged from 7 to 1,150 µg/ml (n=111) and 7 to 3,600 µg/ml (n=270), respectively, and the mean (± standard deviation) concentrations in logarithmic form were 2.23 ± 0.39 and 2.70 ± 0.39, respectively (Tables 1 and 2). The milk Lf concentration in normal lactating cow showed a normal distribution after logarithmic transformation (chi-square test for goodness of fit, p<0.05) (Fig. 1). The 95% confidence range (mean ± 2 standard deviation) for the milk Lf concentration in normal lactating cow is between 27.46 µg/ml and 1,040.10 µg/ml, and the mean concentration is 169.06 µg/ ml. The mean milk Lf concentration (log) in subclinical mastitic cows was significantly (p<0.01) higher than that in normal cows (Tables 1 and 2). Milk Lf concentrations (log) in subclinical mastitic cows classified according to bacterial species are shown in Table 2. The mean Lf concentrations (log) in quarter milk infected with SA and OS were significantly higher than those in quarter milk infected with CNS and CB (p<0.01), respectively, and the mean milk Lf concentrations (log) in quarter milk infected with each of the bacterial species except for that infected with CB were significantly higher than that in normal cows. The mean milk Lf concentration was the lowest for quarter milk infected with CB. Relationship between age of cows and milk Lf concentration: The mean milk Lf concentration (log) in normal lactating cows 5 years of age was significantly lower than those in cows 2 years of age (p<0.01) and 3 years of age (p<0.05) (Table 1). The milk Lf concentration was associated with the age of the cow (one-way analysis of variance, p<0.01). There was a relationship between the milk Lf concentration (log) and the age of the cow (Spearman s correlation coefficient = 0.38, p<0.001). Relationship between lactational stage and milk Lf concentration: Milk Lf concentrations (log) in normal lactating cows classified according to the lactational stage are shown in Table 1. The mean milk Lf concentration (log) in cows in the latter lactational period tended to be higher than that in Table 1. Lactoferrin (Lf) concentrations in quarter milk from normal lactating cows Normal milk Number of samples Milk Lf concentration in logarithmic form mean± standard deviation Total 111 2.23 ± 0.39 2 years 20 2.44 ± 0.30 a) Age of cows 3 years 43 2.30 ± 0.33 a) 4 years 20 2.15 ± 0.40 ab) 5 years 24 2.02 ± 0.49 b) 6 years 4 2.02 ± 0.02 ab) the peak period 8 2.06 ± 0.43 a) Stage of lactation the middle period 59 2.23 ± 0.38 a) the latter period 32 2.30 ± 0.45 a) SCC0 36 2.18 ± 0.19 a) Somatic cell count score SCC1 28 2.16 ± 0.42 a) SCC2 39 2.27 ± 0.51 a) a) and b) values (Mean ± SD) in the same row with different superscripts differ significantly (p<0.05). Somatic cell count score: The somatic cell count score was classified by the number of milk somatic cell as follows. 0 SCC 0 < 18,000 18,000 SCC 1 < 36,000 36,000 SCC 2 < 71,000 Stage of lactation: The stage of lactation was classified as follows the peak period : 50 109 days from parturition the middle period: 110 219 days from parturition the latter period : 220 the end of lactation
MILK LACTOFERRIN CONCENTRATIONS IN NORMAL AND MASTITIC COWS 321 Table 2. Lactoferrin (Lf) concentrations in quarter milk from cows with subclinical mastitis Subclinical mastitis Number of samples Milk Lf concentration in logarithmic form mean ± standard deviation Total 270 2.70 ± 0.39 SA 84 2.83 ± 0.27 a) Bacteria species SAG 8 2.90 ± 0.22 ab) OS 61 2.78 ± 0.39 a) CNS 92 2.58 ± 0.38 bc) CB 25 2.37 ± 0.56 c) SCC 0 8 2.43 ± 0.53 ab) SCC 1 11 2.53 ± 0.37 ab) SCC 2 29 2.41 ± 0.53 a) Somatic cell count score SCC 3 33 2.62 ± 0.29 ab) SCC 4 26 2.73 ± 0.35 ab) SCC 5 10 2.90 ± 0.18 b) SCC 6 6 2.51 ± 0.76 ab) SCC 7 3 3.13 ± 0.22 ab) a),b) and c) values (mean ± standard deviation) in the same row with different superscripts differ significantly (p<0.05). Somatic cell count score: The somatic cell count score was classified by the number of milk somatic cell as follows. 0 SCC 0 < 18,000 283,000 SCC 5 < 566,000 18,000 SCC 1 < 36,000 566,000 SCC 6< 1,132,000 36,000 SCC 2 < 71,000 1,132,000 SCC 7 < 2,263,000 71,000 SCC 3 < 142,000 2,263,000 SCC 8 < 4,536,000 142,000 SCC 4 < 283,000 4,536,000 SCC 9 Fig. 1. Distribution of lactoferrin (Lf) concentration (log) in normal milk samples. cows in the peak or middle period, but there was no clear relationship between the milk Lf concentration and the lactational stage. Relationship between milk SCC score and milk Lf concentration: Milk Lf concentrations (log) in normal lactating cows and in subclinical mastitic cows classified according to the SCC score are shown in Tables 1 and 2, respectively. The milk Lf concentration tended to increase in proportion to the increase in the SCC score. DISCUSSION This study showed that the milk Lf concentration in dairy cows is associated with the age of the cow, but Harmon et al. [7] reported that there is no significant relationship between aging and the measured milk Lf value. This discrepancy in findings might be due to differences in the ages of the dairy cows and amounts of milk production in the two studies: the mean ages of cows in the present study and in the study by Harmon et al. were 3.3 years old and 2.5 years old, respectively, and the mean amounts of milk production
322 S. HAGIWARA, K. KAWAI, A. ANRI AND H. NAGAHATA in the two studies were 29.8 kg and 8.6 kg, respectively. The mean Lf concentration (µg/ml) in the present study is similar to those previously reported [7, 22, 23]. The mean Lf concentration in quarter milk affected with subclinical mastitis was significantly (p<0.001) higher than that in normal milk, as has been reported previously [8]. Lf concentrations in milk infected with major pathogens such as SA and OS were significantly higher than those in milk infected with minor pathogens (i.e., CNS and CB). The Lf concentration in the milk of dairy cows might depend on the pathogenicity of each bacterial species. In the present study, the lowest Lf concentration was found in milk infected with CB, suggesting that CB has a low pathogenicity. The growth of pathogens in the mammary gland must be inhibited to prevent clinical mastitis infection, particularly infection with E. coli, which grows rapidly. A low Lf concentration in milk may lead to rapid growth of E. coli and clinical symptoms as a consequence. It is reported that Lf at a milk concentration of less than 200 µg/ml could not inhibit the growth of E. coli [5, 17]. In the present study, the milk Lf concentration was less than 200 µg/ml in 52% of the normal lactating cows, most of which were aged cows or cows with a low SCC. Cows with a milk Lf concentration of less than 200 µg/ml may not have sufficient ability to inhibit the growth of E. coli. Suriyasathaporn et al. [21] reported that a very low SCC in an udder inflammation-free state is associated with increased risk of clinical mastitis. A low SCC has the same implications as a low Lf concentration, because there is a close correlation between the Lf concentration and SCC, so that a low milk Lf concentration may imply a high risk of clinical mastitis. 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