Assessment of antimicrobial transfer from treated to untreated mammary gland quarters by use of high-pressure liquid chromatography for detection of cloxacillin in milk samples from nonlactating dairy cows

OBJECTIVE: To determine whether half-udder intramammary infusion of cloxacillin results in transfer of cloxacillin from treated to untreated mammary gland quarters within nonlactating cows, and, if so, at what concentrations, and to determine whether selection of ipsilateral versus diagonal-contralateral quarters for treatment affects cloxacillin transfer among quarters. ANIMALS: 20 Holstein-Friesian cows from a dairy herd. PROCEDURES: A within-cow half-udder comparison trial was used in which 2 of 4 mammary gland quarters (ipsilaterally or diagonally) received an intramammary infusion of cloxacillin on day 1 of the nonlactating period. Three days later, milk samples were taken from all untreated quarters and high-pressure liquid chromatography was used to detect and quantify milk cloxacillin concentrations. RESULTS: Cloxacillin was detected in 25% of all untreated mammary gland quarters. Mean cloxacillin concentration in untreated quarters was below minimum inhibitory concentrations for targeted mastitis pathogens. No significant difference in cloxacillin concentrations was found in the ipsilateral or diagonal treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Within-cow half-udder comparison trials are valid for antimicrobial trials in nonlactating cows, although transfer of antimicrobials does occur in trace concentrations. Ipsilateral or diagonal-contralateral treatment designs perform similarly. This type of design is economical for researchers, although care must be taken to account for within-cow clustering of mammary gland quarter data.     

Interleukin-1β infusion in bovine mammary glands prior to challenge with <Emphasis Type="Italic">Streptococcus uberis</Emphasis> reduces bacterial growth but causes sterile mastitis

Dairy cows are especially vulnerable to intramammary infection by the bacterial pathogen Streptococcus uberis in the dry period. Use of immunotherapeutic agents at drying off could increase cellular defences in the gland and prevent establishment of new S. uberis infections. This study investigated the potential of infusing recombinant bovine interleukin-1 beta (rbIL-1beta) in the mammary glands as a prophylactic agent against subsequent intramammary challenge with S. uberis in the early dry period. Immediately after the last milking at commencement of the dry period, one cow from each of 10 monozygous twinsets was infused with 10 microg of rbIL-1beta in two quarters and the other twin was infused with the carrier agent, sterile phosphate buffered saline. Twenty-four hours later, the quarters were infused with 10(3) colony-forming units (CFU) of S. uberis. Bacteriology, somatic cell count (SCC), concentrations of specific cytokines and antibody responses were monitored in mammary gland secretions and sera for the next 21 days. Infusion of rbIL-1beta into mammary glands at commencement of the dry period was associated with less new S. uberis intramammary infections, as determined by the number of quarters with bacterial growth. However, high SCC in quarters following infusion of rbIL-1beta masked the full beneficial effect of this procedure.     

Experimental intramammary infection of the dairy cow with Escherichia coli during the nonlactating period

The nonlactating mammary gland was experimentally inoculated with Escherichia coli. During the first half of the nonlactating period, 32% of 34 inoculated glands were temporarily infected. All intramammary infections were eradicated by the cow without therapy and no signs of mastitis were observed. During the 30 days before parturition occurred, 88% of 42 inoculated glands in the cows became infected. Twenty-three intramammary infections were eradicated by the cow and infection in 14 glands persisted after parturition occurred. Peracute toxic mastitis occurred in those cows with infected glands.     

Function of phagocytes obtained from lacteal secretions of lactating and nonlactating cows

Phagocytes, macrophages and neutrophils, were obtained from lacteal secretions of lactating (n = 13) and nonlactating cows (n = 14). Secretions from nonlactating cows were collected at 7 and 14 days after cessation of lactation. Phagocytes were incubated in vitro with Staphylococcus aureus or Escherichia coli, and function was assessed by fluorescent microscopy of cell suspensions stained with acridine orange and crystal violet. A greater percentage of macrophages from nonlactating cow secretions collected on day 14 phagocytized bacteria than did those collected on day 7. A greater percentage of macrophages from nonlactating cow secretions collected on days 7 and 14 phagocytized bacteria than did neutrophils obtained from the same secretions. A similar percentage of phagocytes from nonlactating cow secretions phagocytized bacteria, compared with phagocytes from lactating cow secretions. Results indicated that the intramammary macrophage may be most important in defense of the mammary gland during the early nonlactating period, because it was more phagocytic than the neutrophil and was more active at 14 days than at 7 days into the nonlactating period.     

Effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period

To evaluate the clinical effects of bovine lactoferrin on staphylococcal mastitis in Holstein cows during the early non-lactating period, 41 mammary quarters were selected randomly from 36 cows on 3 dairy farms. Twelve quarters were infused intramammarily with bovine lactoferrin. Twenty-nine quarters were infused with antibiotic as a control. In the bovine lactoferrin-infused group, 91.7% of mastitic quarters were cured at 7 days after calving, compared with 48.3% in the control group. Furthermore, the changes in mammary secretion induced by the infusion of bovine lactoferrin were investigated. Mean numbers of staphylococci in mammary gland secretions were significantly decreased in both 5 bovine lactoferrin-infused quarters and 5 antibiotic-infused control quarters (p<0.05). Unlike in the control quarters, the mean total cell concentration in the mammary gland secretions increased in bovine lactoferrin-infused quarters. Similar results were obtained in 6 healthy quarters which were infused with bovine lactoferrin. In these quarters, the cell population contained mainly phagocytes such as polymorphonuclear leukocytes and cells positive for CD11b which is known as a complement receptor. The mean concentration of C3 in mammary gland secretions was significantly increased in 5 mastitic quarters infused with bovine lactoferrin (p<0.05), but showed no significant change in 5 mastitic control quarters. These results suggested that bovine lactoferrin treatment for staphylococcal mastitis in the early non-lactating period might increase the rate of cure through the induction of innate immunity in the host.     

Efficacy of extended pirlimycin therapy for treatment of experimentally induced Streptococcus uberis intramammary infections in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, around the time of calving, and during early lactation. Strategies for controlling S. uberis mastitis have not received adequate research attention and are therefore poorly defined and inadequate. Objectives of the present study were to evaluate the efficacy of extended therapy regimens with pirlimycin for treatment of experimentally induced S. uberis intramammary infections in lactating dairy cows during early lactation and to evaluate the usefulness of the S. uberis experimental infection model for evaluating antimicrobial efficacy in dairy cows. The efficacy of extended pirlimycin intramammary therapy regimens was investigated in 103 mammary glands of 68 dairy cows that became infected following experimental challenge with S. uberis during early lactation. Cows infected with S. uberis in one or both experimentally challenged mammary glands were randomly allocated to three groups, representing three different treatment regimens with pirlimycin, including 2-day (n = 21 cows, 31 mammary quarters), 5-day (n = 21 cows, 32 quarters), and 8-day (n = 26 cows, 40 quarters). For all groups, pirlimycin was administered at a rate of 50 mg of pirlimycin hydrochloride via intramammary infusion. A cure was defined as an experimentally infected mammary gland that was treated with pirlimycin and was bacteriologically negative for the presence of S. uberis at 7, 14, 21, and 28 days after treatment. Experimental S. uberis intramammary infections were eliminated in 58.1% of the infected quarters treated with the pirlimycin 2-day regimen, 68.8% for the 5-day regimen, and 80.0% for the 8-day regimen. Significant differences (P <.05) in efficacy were observed between the 2-day and 8-day treatment regimens. The number of somatic cells in milk decreased significantly following therapy in quarters for which treatment was successful in eliminating S. uberis. However, there was no evidence to suggest that extended therapy with pirlimycin resulted in a greater reduction in somatic cell counts in milk than the 2-day treatment. The S. uberis experimental infection model was a rapid and effective means of evaluating antimicrobial efficacy during early lactation at a time when mammary glands are highly susceptible to S. uberis intramammary infection.     

Intramammary infusion of an Enterococcus faecium SF68 preparation promoted the involution of drying off Holstein cows partly related to neutrophil‐associated matrix metalloproteinase 9

A problem for dairy cows following milk stasis is to cope with a high risk of intramammary infection and there is a need to initiate an extensive renewal of secretory modules in mammary glands so that milk production in next lactation may be optimized. We recently reported that ultrasonicated Enterococcus faecium SF68 (SF68) is compatible with cow mammary glands and an enhancer of innate immunity during the immediate post‐milk stasis period. The current study further examines the concomitant effect of ultrasonicated SF68 on mammary tissue remodeling. Four Holstein cows each received intramammary infusions of regular antibiotic dry‐cow formula (positive control) and two different doses of SF68 in different quarters. Analyses of individual quarter secretion samples showed faster neutrophil infiltration, earlier modifications in protein composition, including caseins and lactoferrins, as well as more prompt elevation of the specific unit of 92‐kDa matrix metalloproteinase 9 (MMP9) in SF68‐infused quarters compared to the positive controls. Intramammary infusion of ultrasonicated SF68 seems able to accelerate the regression of mammary synthetic capacity and potentiate the breakdown of glandular extracellular matrix, indicating a more efficient mammary gland involution. Correlation analyses imply that the ability of ultrasonicated SF68 to induce faster neutrophil chemotaxis and the associated MMP9 release is partly responsible.     

Experimental infection of bovine mammary glands with Streptococcus agalactiae during the nonlactating period

Two experiments were done to determine the rate of intramammary infection (IMI) in dairy cows during the nonlactating period. In experiment 1, all glands were equally exposed to Klebsiella pneumoniae and Streptococcus agalactiae for the first 3 weeks after the start of the nonlactating period. Nearly all new IMI was caused by S agalactiae. In experiment 2, the susceptibility of nonlactating mammary glands to new IMI by S agalactiae after direct inoculation into the lactiferous sinus was determined. During the 1st month of the nonlactating period, 30% of inoculated glands became infected. During the last month of the nonlactating period, 90% of inoculated glands became infected.     

Tuberculin elicited cellular immune response in the lactating bovine mammary gland vaccinated intramammarily with Mycobacterium bovis

The development of a local antigen-specific sensitivity was monitored histologically and in secretions of the bovine mammary gland. Three cows in mid-lactation were immunized by injecting 50 microliter of a killed Mycobacterium bovis vaccine into the dorsal secretory tissue of the rear mammary glands; two cows served as unvaccinated controls. Ten weeks after vaccination, all cows were challenged by intramammary infusion of 1.0 microgram tuberculin in 5 ml phosphate-buffered saline (PBS). Three quarters of each cow received tuberculin at 72, 48, and 24 hours before slaughter; a control quarter received PBS at 72 hours. Vaccinated cows exhibited an intense, local cellular reaction to tuberculin in teat-end tissues at all times post infusion; PBS-infused glands were normal. A moderate leukocyte response in parenchymal tissues adjacent to the gland cistern of tuberculin-infused quarters was observed, but deep parenchymal tissues were normal; no effect on milk yield was found. Tuberculin-infused quarters exhibited histological responses in teat cisternal tissues similar to those in delayed-type hypersensitivity reactions. Leukocytic accumulation was primarily macrophages and lymphocytes with few neutrophils. Erythema was observed in the distal half of the cistern, and fibrin deposits were found in subepithelial connective tissues. The epithelium, although distended with leukocytes, was intact and numerous mitotic figures were present. Unvaccinated cows showed no response to challenge. Results demonstrated a marked and specific cellular response in sensitized cows to challenge with tuberculin.     

Growth of Staphylococcus aureus and Streptococcus species in bovine mammary secretions during the nonlactating and peripartum periods following intramammary infusion of lipopolysaccharide at cessation of milking.

The objective was to determine if induced mammary inflammation at cessation of milking influenced growth of gram-positive mastitis pathogens in mammary secretions, particularly during early involution. Growth of all mastitis pathogens evaluated was similar in cell-free fat-free mammary secretions from LPS-infused and control glands. These data indicate that intramammary infusion of LPS at cessation of milking did not alter growth of gram-positive mastitis pathogens in mammary secretion during the nonlactating period. Stage of lactation and the nonlactating period influenced bacterial growth and marked differences between bacteria and among strains of a bacterial species were observed. Staphylococcus aureus grew well in secretions collected during late lactation, but growth decreased during early- and mid-involution and increased again in secretions obtained near parturition. Streptococcus agalactiae and Strep. uberis grew better in mammary secretion obtained during involution than in secretions collected during late or early lactation. Streptococcus dysgalactiae grew well in mammary secretions at all time periods. These data demonstrate the variability of mastitis pathogen growth during physiologic transitions of the bovine udder.     

Effects of antibiotics on phagocyte recruitment, function, and morphology in the bovine mammary gland during the early nonlactating period

The effects of 2 antibiotic preparations administered intramammarily on phagocyte recruitment, function, and morphology were evaluated at the beginning of the nonlactating period. Twelve cows with no clinical or microbiologic evidence of mastitis were assigned to 1 of 2 treatment groups. At the end of lactation, 1 of the antibiotic preparations was infused in a fore- and hind quarter of each cow; the remaining quarters were untreated controls. One group was given benzathine cephapirin; the second group was given sodium novobiocin. Secretion samples were collected from 1 treated and 1 control quarter at 16 hours, and from the remaining 2 quarters at 64 hours after treatment. Total and differential somatic cell counts were determined, and morphology of mammary polymorphonuclear neutrophils (PMN) and macrophages was observed by transmission electron microscopy. In vitro ingestion and killing of Staphylococcus aureus by mammary PMN and macrophages were assessed by fluorescent microscopy, using acridine orange stain. Cells resident in a fixed volume of secretion were incubated with a known concentration of S aureus. Total cell and PMN concentrations were higher in treated than in control quarters. Neutrophils were the predominant cell type in both treated and control quarters over the sampling period. As measured in this study, in vitro ingestion and killing of S aureus by individual PMN from treated quarters was reduced. Antibiotic treatment also increased the proportion of morphologically abnormal phagocytes. There were significant correlations among PMN ingestion, killing, and morphology. However, increased PMN concentrations tended to compensate for the reduced phagocytic function of individual cells. Therefore, efficacy of antibiotic treatment of nonlactating cows may depend, at least in part, on increased PMN concentration, which may tend to compensate for reduced phagocytic function. Compared with PMN, macrophages appeared to have only a minor role in phagocytosis of bacteria.     

Frequency of isolation of environmental mastitis-causing pathogens and incidence of new intramammary infection during the nonlactating period

Quarter samples (n = 6,328) of mammary secretions were collected from 160 cows during physiologic transitions of the udder to determine the frequency of isolation of mastitis-causing pathogens and the incidence of new intramammary infections (IMI) during the nonlactating period. None of the cows in the herd was infected with Streptococcus agalactiae, and the prevalence of Staphylococcus aureus was low. Cows were not treated with antibiotics at cessation of milking. A threefold increase in the percentage of quarters infected with major mastitis-causing pathogens developed from late lactation to early involution. Coliforms and streptococci other than Str agalactiae accounted for 94% of major pathogen infections. The number of quarters infected with coagulase-negative staphylococci increased slightly from late lactation to early involution, whereas the number of quarters infected with Corynebacterium bovis decreased markedly. Major pathogens caused 101 of 153 IMI at parturition and greater than 90% were caused by streptococci and coliforms. At parturition, 51 of 52 minor pathogen IMI were caused by coagulase-negative staphylococci. During early lactation, there was a marked decrease in quarters infected with major pathogens; however, the number of quarters with major pathogen IMI during early lactation was 2.3 times higher than the number of quarters infected before cessation of milking. The number of quarters with minor pathogen IMI during early lactation was the same as at parturition, but a marked decrease in quarters infected with coagulase-negative staphylococci and a marked increase in C bovis IMI developed from parturition to early lactation.     

Gram-negative bacterial infections of the mammary gland in cows

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were less than 28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P less than 0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.     

Elimination kinetics of ceftiofur hydrochloride after intramammary administration in lactating dairy cows

OBJECTIVE: To determine the elimination kinetics of ceftiofur hydrochloride in milk after intramammary administration in lactating dairy cows. DESIGN: Prospective study. ANIMALS: 5 lactating dairy cows. PROCEDURE: After collection of baseline milk samples, 300 mg (6 mL) of ceftiofur was infused into the left front and right rear mammary gland quarters of each cow. Approximately 12 hours later, an additional 300 mg of ceftiofur was administered into the same mammary gland quarters after milking. Milk samples were collected from each mammary gland quarter every 12 hours for 10 days. Concentrations of ceftiofur and its metabolites in each milk sample were determined to assess the rate of ceftiofur elimination. RESULTS: Although there were considerable variations among mammary gland quarters and individual cows, ceftiofur concentrations in milk from all treated mammary gland quarters were less than the tolerance (0.1 microg/mL) set by the FDA by 168 hours (7 days) after the last intramammary administration of ceftiofur. No drug concentrations were detected in milk samples beyond this period. Ceftiofur was not detected in any milk samples from nontreated mammary gland quarters throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Ceftiofur administered by the intramammary route as an extra-label treatment for mastitis in dairy cows reaches concentrations in milk greater than the tolerance set by the FDA. Results indicated that milk from treated mammary gland quarters should be discarded for a minimum of 7 days after intramammary administration of ceftiofur. Elimination of ceftiofur may be correlated with milk production, and cows producing smaller volumes of milk may have prolonged withdrawal times.     

Efficacy of using an internal teat sealer to prevent new intramammary infections in nonlactating dairy cattle

OBJECTIVE: To determine whether insertion of an internal teat sealer (ITS) at the end of lactation would prevent development of new intramammary infections (IMIs) during the nonlactating period. DESIGN: Controlled clinical trial. ANIMALS: 939 Holstein-Friesian dairy cows from 16 herds. PROCEDURES: Results of bacteriologic culture of milk samples collected 14 days prior to the end of lactation were used to assign cows to groups (group 1 = negative results for all quarters; group 2 = positive results for > or = 1 quarter). Quarters of cows in group 1 were treated with an ITS or a single intramammary dose of cloxacillin; quarters of cows in group 2 were treated with cloxacillin in conjunction with an ITS or with cloxacillin alone. Milk samples were collected at the end of lactation and within 8 days after calving. RESULTS: Regardless of whether the outcome of interest was new IMIs caused by any pathogens, major pathogens, environmental pathogens, or streptococci other than Streptococcus agalactiae, quarters in group 2 treated with both cloxacillin and an ITS were less likely to develop a new IMI than were quarters treated with cloxacillin alone. For cows in group 1, no significant difference in risk of new IMIs was found between treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for dairy cattle with an IMI late in the lactation period, intramammary administration of cloxacillin at the end of lactation followed by insertion of an ITS enhanced protection against development of new IMIs, compared with use of cloxacillin alone.     

Effects of intramammary infusions of interleukin-8 on milk protein composition and induction of acute-phase protein in cows during mammary involution

The effects of interleukin-8 (IL-8) on bovine mammary functions such as milk protein secretion and the blood-milk barrier during mammary involution were evaluated. Following the final milking, recombinant bovine (rb) IL-8 (5 or 25 microg) and a saline placebo were individually infused into the left- and right-front teat cisterns of 6 cows, respectively. Three cows without treatment at the final milking were also used as controls. Mammary secretions and blood were collected at -24, 0, 10, 24, 72, 168, 336, and 720 h after infusion. In the mammary glands infused with 25 microg of rbIL-8, the increases in somatic cell counts and in the concentrations of serum albumin, IgG1 and IgG2, and the decreases in the concentrations of alpha- and beta-casein and beta-lactoglobulin were greater than in the control glands. In the mammary glands infused with 5 microg of rbIL-8, compared to the glands infused with 25 microg of rbIL-8, these changes were moderate. These results indicate that rbIL-8 impairs the integrity of the blood-milk barrier and suppresses milk-specific protein secretions. In the cows infused with 25 microg of rbIL-8, the rectal temperature and serum haptoglobin level were transiently elevated after the infusion, showing that intramammary infusion of rbIL-8 could elicit systemic inflammation.     

Growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution of the bovine mammary gland: relation to secretion composition

Mammary secretions from 12 Holstein dairy cows were collected to evaluate growth inhibition of Escherichia coli and Klebsiella pneumoniae during involution and during physiologic transitions of the mammary gland. Mammary secretions obtained during late lactation poorly inhibited growth of E coli and K pneumoniae. However, as involution progressed, mammary secretions increasingly inhibited growth of both coliform mastitis pathogens. Greatest inhibition of E coli and K pneumoniae growth was observed when mammary glands were fully involuted. Growth inhibition remained high until 7 days before parturition, and then it decreased significantly (P less than 0.05) to that observed during late lactation. Inhibition of coliform mastitis pathogen growth was associated with high concentrations of lactoferrin and immunoglobulin G, decreased citrate concentration, and a low citrate to lactoferrin molar ratio. These data suggested that differences in susceptibility or resistance to new intramammary infection with coliform mastitis pathogens during the nonlactating period may be attributable, in part, to marked changes in mammary secretion composition that develop during physiologic transitions of the mammary gland. Resistance of the fully involuted mammary gland to coliform infection may be associated with high concentrations of natural protective factors.     

Resistance mechanisms of the bovine udder: new implications for mastitis control at the teat end

Investigations on bovine teat-end defenses and their role in mastitis control were reviewed. Alteration of teat canal keratin by method of intramammary drug infusion through the teat canal influenced the number of new infections. At the beginning of the nonlactating period, 2 methods of administering antibiotic were studied: full insertion of treatment syringe cannulas into teat cisterns and expelling contents and partial insertion of cannulas into the distal 2 to 3 mm of teat canals and slowly infusing contents. Partial cannula insertion reduced new infections by 50% and is an easily adopted management tool to reduce prevalence of mastitis. Intramammary devices used during lactation and the nonlactating period increased leukocyte concentrations in mammary secretions. An abraded polyethylene coil device reduced clinical mastitis and increased milk yield. Staphylococci colonize teat canal keratin and lacteal secretions of dairy heifers as early as 9 months of age, leading to intramammary infection at time of calving and persisting into lactation. Subsequent somatic cell counts are associated with milk production losses. Previously, such infections were shown to be associated only with older, mature animals.     

Accelerated bovine mammary involution induced by infusion of concanavalin A or phytohemagglutinin

Right mammary quarters of dairy cows were infused with concanavalin A (conA) or phytohemagglutinin (PHA) near drying off. Significant changes in milk yield and composition were observed by 24 hours after infusion. In general, conA or PHA treatment resulted in significantly greater compositional changes earlier in the dry period compared with control quarters. As involution progressed, lactoferrin, serum albumin, immunoglobulin G, pH, and numbers of somatic cells increased, whereas citrate and the molar ratio of citrate:lactoferrin decreased. Mammary secretion accumulation at 7 days of involution was significantly lower in treated compared with control quarters. The data indicated that intramammary infusion of conA or PHA near drying off accelerates bovine mammary involution, resulting in elevated levels of natural protective factors.     

Efficacy of intramammary infusion of ceftiofur hydrochloride at drying off for treatment and prevention of bovine mastitis during the nonlactating period

This study evaluated the efficacy of intramammary infusion of ceftiofur hydrochloride for the treatment of intramammary infections present at the last milking of lactation and for prevention of new intramammary infections during the nonlactating period. Cows were randomly assigned to five treatments (untreated negative control, 125, 250, and 500 mg of ceftiofur, and a positive control group receiving 300 mg cephapirin benzathine). A dose of 125 mg of ceftiofur per mammary quarter was effective for treatment of existing infections present at the time of milk cessation, but only the 500-mg dose of ceftiofur per mammary quarter was effective for both treatment of existing intramammary infections at the time of milk cessation and for prevention of new intramammary infections during the nonlactating period.