Growth of Gram-Negative Bacteria in Dry Cow Secretion 1 DEBORAH TODHUNTER,2 K. LARRY SMrTH,2,3 and JOSEPH S. HOGAN2 The Ohio State University Ohio Agricultural Research and Development Center Wooster 44691 ABSTRACT
Gram-negative bacteria (n = 192) isolated from infected bovine mammary glands were tested for growth in a pooled source of dry cow secretion. Growth of Klebsiella pneumoniae in dry cow secretion was greater than growth of Escherichia coli and Klebsiella oxytoca. Escherichia coli originating during the early dry period exhibited greater growth in dry cow secretion than those originating around calving or during lactation. Klebsiella pneumoniae growth did not differ with time of origin of intrarnammary infection. Escherichia coli. K. oxytoca, and K. pneumoniae growth in a synthetic medium was reduced by apolactoferrin plus Ig. Growth reduction was greatest for E. coli. Otrate reversed growth inhibition. The inhibitory properties of dry cow secretion for E. coli may contribute to the low number of naturally occurring intramammary infections originating during the early part of the dry period. Inhibitory properties of dry cow secretion are partially explained by laetoferrin acting in conjunction with antibody to prevent iron acquisition by many gram-negative bacteria. (Key words: gram-negative bacteria, dry cow secretion. lactoferrin) INTRODUCTION
The rate of bovine gram-negative bacterial intramammary infection (IMI) is three- to fourfold greater during the dry period than during
Received May 15. 1989. Accepted August 24. 1989. ISalaries and research suppon provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 133-89. 2Departrnent of Dairy Science. 3Departrnent of Vetrinary Science. 1990 J Dairy Sci 73:363-372
lactation (22). Rate of infection is not constant across the dry period, but rather, it is elevated during the fITst 2 wk of the dry period and the 2 wk prior to parturition. Studies on naturally occurring IMI indicate that the majority of Escherichia coli IMI associated with the dry period are fITst detected immediately prior to or at parturition (23; Todhunter et al.. unpublished). In contrast, origin of Klebsiella spp. IMI was equally divided between the fITst and second halves of the dry period. Gram-negative bacteria require iron for growth and possess several genetically controlled high affinity acquisition systems to obtain iron in iron-limiting environments. Two of these iron acquisition systems involve the production of low molecular weight iron chelators (siderophores) and outer membrane protein receptors for the iron-laden siderophore (15). Gram-negative bacteria also can utilize citrate to obtain iron; however, citrate must be present for the induction of the citrate iron acquisition system (10. 12). Due to the high concentration of lactoferrin (Lf) and low concentration of citrate in the involuted gland. gram-negative bacteria would be genetically forced to utilize an iron acquisition system other than citrate during involution. At parturition and during lactation. gram-negative bacteria could potentially utilize the citrate iron acquisition system due to the high concentration of citrate in bovine milk and colostrum (16. 27). Immunoglobulin enhances the bacteriostatic properties of Lf for gram-negative bacteria (6. 25. 26). The finding that the involuting gland is relatively resistant to experimental and natural infection with E. coli indicates that antibody is present in mammary secretion than can prevent E. coli from acquiring iron with a siderophore iron acquisition system. Additionally. the antibody may not be as effective against other gram-negative bacteria that establish infection during the first half of the dry period. such as Klebsiella spp. The objectives of this study were to compare growth of different species of
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gram-negative bacteria in secretion obtained from the involuted gland and in defined semisynthetic medium containing Lf and Ig. MATERIALS AND METHODS Animals, Sampling and Microbiological Procedures, and Infection History Parameters
Experimental animals were Holstein (80%) and Jersey (20%) dairy cattle from the Krauss Dairy Center of The Ohio Agricultural Research and Development Center (OARDC), Wooster. Herd size averaged 150 lactating animals. Housing and management of cows, quarter milk sample collection, and microbiological procedures were as described previously (22). Quarter foremilk samples for microbiological culture were obtained from all cows at the following times: 14 and 7 d prior to drying off; at drying off (prior to intramammary infusion of antibiotics); 30 d of the dry period; calving; 3, 7, 14, and 30 d postealving; and at 3D-d intervals for the remainder of lactation. Single quarter milk samples were obtained at all time points except at 30 d of involution when samples were obtained in duplicate. Diagnosis and Origin by Stage of Lact8Uon of Gram-Negative Bacterial Intramammary Infections
A gram-negative IMI was diagnosed when one of the following criteria was met: 1) single isolation of a pathogen from a clinical quarter; 2) same pathogen was isolated from both duplicate quarter samples; or 3) same pathogen was isolated from two out of three consecutive samples with less than 31 d between isolations. An exception to the 30 d between isolations was when the same gram-negative bacterial species was isolated at 30 d of the dry period and again at calving and when greater than 30 d had elapsed between calving and the 30-
Gram-negative bacteria (n = 192) isolated from infected bovine mammary glands were tested for growth in a pooled source of dry cow secretion. Growth of Klebsiella pneumoniae in dry cow secretion was greater than growth of Escherichia coli and Klebsiella oxytoca. Escherichia coli originating during the early dry period exhibited greater growth in dry cow secretion than those originating around calving or during lactation. Klebsiella pneumoniae growth did not differ with time of origin of intrarnammary infection. Escherichia coli. K. oxytoca, and K. pneumoniae growth in a synthetic medium was reduced by apolactoferrin plus Ig. Growth reduction was greatest for E. coli. Otrate reversed growth inhibition. The inhibitory properties of dry cow secretion for E. coli may contribute to the low number of naturally occurring intramammary infections originating during the early part of the dry period. Inhibitory properties of dry cow secretion are partially explained by laetoferrin acting in conjunction with antibody to prevent iron acquisition by many gram-negative bacteria. (Key words: gram-negative bacteria, dry cow secretion. lactoferrin) INTRODUCTION
The rate of bovine gram-negative bacterial intramammary infection (IMI) is three- to fourfold greater during the dry period than during
Received May 15. 1989. Accepted August 24. 1989. ISalaries and research suppon provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 133-89. 2Departrnent of Dairy Science. 3Departrnent of Vetrinary Science. 1990 J Dairy Sci 73:363-372
lactation (22). Rate of infection is not constant across the dry period, but rather, it is elevated during the fITst 2 wk of the dry period and the 2 wk prior to parturition. Studies on naturally occurring IMI indicate that the majority of Escherichia coli IMI associated with the dry period are fITst detected immediately prior to or at parturition (23; Todhunter et al.. unpublished). In contrast, origin of Klebsiella spp. IMI was equally divided between the fITst and second halves of the dry period. Gram-negative bacteria require iron for growth and possess several genetically controlled high affinity acquisition systems to obtain iron in iron-limiting environments. Two of these iron acquisition systems involve the production of low molecular weight iron chelators (siderophores) and outer membrane protein receptors for the iron-laden siderophore (15). Gram-negative bacteria also can utilize citrate to obtain iron; however, citrate must be present for the induction of the citrate iron acquisition system (10. 12). Due to the high concentration of lactoferrin (Lf) and low concentration of citrate in the involuted gland. gram-negative bacteria would be genetically forced to utilize an iron acquisition system other than citrate during involution. At parturition and during lactation. gram-negative bacteria could potentially utilize the citrate iron acquisition system due to the high concentration of citrate in bovine milk and colostrum (16. 27). Immunoglobulin enhances the bacteriostatic properties of Lf for gram-negative bacteria (6. 25. 26). The finding that the involuting gland is relatively resistant to experimental and natural infection with E. coli indicates that antibody is present in mammary secretion than can prevent E. coli from acquiring iron with a siderophore iron acquisition system. Additionally. the antibody may not be as effective against other gram-negative bacteria that establish infection during the first half of the dry period. such as Klebsiella spp. The objectives of this study were to compare growth of different species of
363
364
TODHUNTER ET AL.
gram-negative bacteria in secretion obtained from the involuted gland and in defined semisynthetic medium containing Lf and Ig. MATERIALS AND METHODS Animals, Sampling and Microbiological Procedures, and Infection History Parameters
Experimental animals were Holstein (80%) and Jersey (20%) dairy cattle from the Krauss Dairy Center of The Ohio Agricultural Research and Development Center (OARDC), Wooster. Herd size averaged 150 lactating animals. Housing and management of cows, quarter milk sample collection, and microbiological procedures were as described previously (22). Quarter foremilk samples for microbiological culture were obtained from all cows at the following times: 14 and 7 d prior to drying off; at drying off (prior to intramammary infusion of antibiotics); 30 d of the dry period; calving; 3, 7, 14, and 30 d postealving; and at 3D-d intervals for the remainder of lactation. Single quarter milk samples were obtained at all time points except at 30 d of involution when samples were obtained in duplicate. Diagnosis and Origin by Stage of Lact8Uon of Gram-Negative Bacterial Intramammary Infections
A gram-negative IMI was diagnosed when one of the following criteria was met: 1) single isolation of a pathogen from a clinical quarter; 2) same pathogen was isolated from both duplicate quarter samples; or 3) same pathogen was isolated from two out of three consecutive samples with less than 31 d between isolations. An exception to the 30 d between isolations was when the same gram-negative bacterial species was isolated at 30 d of the dry period and again at calving and when greater than 30 d had elapsed between calving and the 30-
