Changes in blood and milk lymphocyte sub-populations during acute and chronic phases of Staphylococcus aureus induced bovine mastitis

Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.     

Staphylococcus aureus exosecretions and bovine mastitis

The role of Staphylococcus aureus and coagulase-negative staphylococcal exosecretions in bovine udder infection was tested by monitoring the cows' response to in vivo inoculation of bacterial exosecretions into udder quarters. Twenty Israeli-Holstein dairy cows were included in the study; two or three of the udder quarters of each cow were intracisternally inoculated with 0.04-0.05 mg/quarter (total proteins) of the various sterile bacterial exosecretions in a sterile pyrogen-free saline. Each udder was inoculated with two or three different bacterial exosecretions or placebo (Columbia Broth). Cows were monitored for 96 h post-inoculation for rectal temperature, heart and respiratory rates, alimentary tract activity (rumen contraction), udder temperature, pain, oedema and udder size. Milk samples were examined bacteriologically and for somatic cell count, N-acetyl-D-glucosaminidase (NAGase) activity and somatic cell differentiation. No enterotoxins (beta-G) or toxic shock syndrome toxin-1 were detected in response to any of the bacteria tested. Control quarters or those inoculated with Columbia Broth, showed similar NAGase and somatic cell count values throughout the experiment. Twelve of the 18 strains tested, induced inflammation in the inoculated quarters while six did not. Of the 12 strains causing local inflammation, only six were found significantly different from the control and were considered as high response (group 1). The other six that caused a local inflammation did not differ significantly from the control, and were considered to be moderate response (group 2). The six S. aureus isolates that did not cause an inflammatory response were considered to have low response (group 3). In all quarters inoculated with S. aureus bacterial exosecretions belonging to groups 1 and 2, the polymorphonuclear cells and macrophages were proportionally increased while CD4+ and CD8+ T-lymphocyte populations decreased. One-dimensional NuPAGE (7%) Tris-acetate gel electrophoresisof the bacterial exosecretions revealed four different bands appearing between 36 and 31 kDa, marked from top to bottom as A, B, C and D. An association was found between the combinations of expressed bands and the cow responses: the majority of the cases could be linked to the expression of bands B and C.     

The effect of experimental infectious mastitis on leukocyte subpopulations and cytokine production in non-lactating ewes.

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Impact of intramammary infections in dairy heifers on future udder health, milk production, and culling

Dairy heifers represent the future of a dairy herd, and are expected to freshen with a healthy and well-developed udder, capable of producing an optimal amount of high quality milk. A high proportion of heifers have infected mammary quarters at calving, with coagulase-negative staphylococci (CNS) being the most common cause. Staphylococcus aureus and environmental pathogens are also found. The aim of this paper is to summarize how intramammary infections during (late) gestation and early lactation impair the development of the mammary gland and negatively affect future udder health and milk production. Heifers calving with either subclinical or clinical mastitis are also at a higher risk to be culled in first lactation. The magnitude of the effect is most likely related to the virulence of the causative pathogen, the persistence of the infection when milk production has started, and the time of onset of infection. Histological changes in udder tissue from quarters infected with S. aureus are more pronounced than those in udder tissue from CNS-infected quarters. The longer the infections exist and the longer they persist into lactation, the larger the impact on heifers' future udder health and milk production will be. In general, CNS infections are cleared early in lactation and some studies show that CNS do not have a large impact on future milk production and udder health. Future research should elucidate to what extent pathogen-specific as well as host-related factors affect the persistence of IMI in early lactating heifers.     

Intramammary infusion of beta1,3-glucan for prevention and treatment of Staphylococcus aureus mastitis

Udder health problems associated with Staphylococcus aureus infections in dairy cows are difficult to control and antibiotics have limited effects. Lately, more interest has been directed towards ways to stimulate the innate immune mechanisms of the animal for better prevention and treatment of mastitis. The objectives of this study were to investigate if intramammary infusion at drying off with the immune modulator beta1,3-glucan can make the udder more resistant to experimental intra mammary S. aureus infection at this time, and to study if intramammary infusion of beta1,3-glucan into lactating udder quarters with chronic subclinical S. aureus infection can stimulate the clearing of the infection. Another aim was to evaluate the effect of beta1,3-glucan on the expression of major histocompatibility complex (MHC class II) on mammary leucocytes, measured by flow cytometry, during these circumstances. The results indicated a slight, but not statistically significant, positive effect of beta1,3-glucan at drying off on the clinical and anti-bacterial response to S. aureus infection, but no therapeutic effect of beta1,3-glucan treatment of udder quarters with chronic subclinical S. aureus mastitis. However, the proportion of MHCII+ milk lymphocytes tended to increase after glucan infusion in those udder quarters indicating a stimulation of the antigen presenting ability. To further evaluate a possible preventive effect of beta1,3-glucan infusion at drying off more studies are needed involving a larger number of animals.     

Characterization of cytokine expression in milk somatic cells during intramammary infections with Escherichia coli or Staphylococcus aureus by real-time PCR

The expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma, by milk somatic cells was characterized by real-time polymerase chain reaction (PCR) in dairy cows experimentally challenged with either E. coli (n = 8) or S. aureus (n = 8). The mRNA abundance of a target gene was calibrated with that of a reference gene (beta-actin) and expressed as fold of induction over the control quarter at each time point. At no single time point did all eight quarters challenged with the same type of bacteria demonstrated increased expression of a target gene and there was large variation among animals at each given time. As a consequence, most tested comparisons were not statistically significant except the peak time points of IL-8 expression (75- and 29- fold in glands challenged with E. coli and S. aureus, respectively). However, the average fold induction of all targeted cytokines was increased in response to both bacterial challenges with the exception of IFN-gamma. The expression of IFN-gamma was only increased in milk somatic cells isolated from E. coli, but not S. aureus, challenged mammary glands. Moreover, upregulated expression of cytokine genes had higher magnitudes and/or faster responses in glands challenged with E. coli in comparison with those challenged with S. aureus. We propose that the compromised upregulation of inflammatory cytokines in S. aureus infected glands may, at least partially, contribute to the chronic course of infection caused by this pathogen. Further research on identifying factors responsible for the differentially expressed cytokine profiles may be fundamental to developing strategies that mitigate the outcome of bovine mastitis.     

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.     

Physiological and morphological changes in bovine mammary glands following intramammary infusion of recombinant interferon-gamma.

Eleven lactating dairy cows were used to evaluate the response of bovine mammary glands to increasing doses of recombinant bovine interferon (rBoIFN)-gamma. Right front and rear quarters were intramammarily infused with eight different doses (10(2) U to 2 x 10(8) U/quarter) of rBoIFN-gamma; each dose was tested in at least two quarters. Left udder halves served as within animal controls in which quarters were injected with a saline placebo or were not infused at all. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 6, 24, 36 and 48 h following infusion. Animals were slaughtered immediately following the 48 h sampling period and mammary tissue was obtained for morphometric analyses. Milk composition was similar between control quarters and those quarters infused with up to 10(5) U of rBo-IFN-gamma during the entire sampling period. Quarters infused with 10(6) U and 10(7) U of rBoIFN-gamma had higher milk somatic cell counts (SCC) following treatment compared with preinfusion values. Changes in the composition of mammary secretion were most dramatic in quarters infused with greater than or equal to 10(8) U of rBoIFN-gamma as indicated by the significant increase in SCC and milk pH with a concomitant decrease in lactose concentration when compared with pre-infusion values or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in luminal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of rBoIFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection.

Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration. Intraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery of S. aureus, but did not eliminate infections. Intravenous injection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily.     

Effect on outcome of intramammary challenge exposure with Staphylococcus aureus of somatic cell concentration and presence of an intramammary device

Results of experimental Staphylococcus aureus intramammary challenge of all quarters of 6 cows, each fitted with an intramammary device (IMD) in 2 quarters, and of 10 quarters of 3 cows not fitted with an IMD were reported. Infection was established in all 34 quarters, regardless of presence or absence of an IMD. Neither the course nor severity of early S aureus intramammary infection were influenced by the presence of an IMD or by differences in milk somatic cell (MSC) concentration in the gland at the time of bacterial challenge infusion, up to a MSC concentration of nearly 1 million/ml. Cumulative success of experimental infection in this and a previous study from our laboratory was nearly 100% in glands in which the MSC concentration was less than 1 million/ml and about 17% when the MSC concentration exceeded 1 million/ml.     

Udder shape and teat-end lesions as potential risk factors for high somatic cell counts and intra-mammary infections in dairy cows

The association of common bacterial pathogens in milk samples during calving with udder shape or the presence of 'teat-end' lesions was investigated in 240 dairy cows from two herds. Sixty-three of 120 cows (53%) in one herd (herd A) and 54/120 animals (45%) in a second herd (herd B) had normal-shaped udders. The remaining animals had udder shapes defined as follows: large pendulous (18% herd A, 26% herd B); large between hindquarter (10% herd A, 17% herd B); overall small (8% herd A, 5% herd B); or small but pendulous (11% herd A, 7% herd B). At calving teat-end lesions were present in 63% and 76% of the quarters of herd A and B animals, respectively. There was no herd effect on udder shape or teat-end lesions. Analysis of variance revealed that udder shape and teat-end lesions did not have a significant association with quarter somatic cell count. However there was some association between mammary infection and udder shape and teat-end lesions. Compared to other udder shapes, cows with large between hindquarter shape had significantly less Staphylococcus aureus and Streptococcus uberis infection (P<0.001). There was a similar albeit less significant negative association with Escherichia coli infection (P<0.01). Infection with Streptococcus agalactiae and Streptococcus dysgalactiae was more frequent in cows with large pendulous and overall small udder conformations. The results also suggest an association between intra-mammary infection at calving and the presence of hyperkeratotic teat-end lesions, given that S. aureus, coagulase-negative staphylococci, S. uberis, S. agalactiae and E. coli were cultured from significantly more quarters with such lesions than from quarters without lesions or with other types of lesion (P<0.001).     

Inflammatory reactions in the teat and udder of the dry cow

The inflammatory reactions in the teat and udder of the dry cow were studied by total and differential somatic cell counts (SCC) and by measuring bovine serum albumin, N-acetyl-beta-D-glucosaminidase (NAGase), plasminogen and plasmin. The teat and udder cisterns were surgically separated from each other in two udder quarters of each cow. Salmonella endotoxin was infused in one teat cistern and one udder quarter, and saline was infused in one teat cistern and one udder quarter. The inflammatory response was followed by several samplings post infusion. The reactions in the dry udder quarters were mainly in line with the results of similar studies in lactating glands. The differential SCC and the NAGase results were, however, somewhat different. The teats were capable of a strong inflammatory response. Reactions different from those seen in the glands were observed with regard to permeability changes and NAGase. The experimental model used showed promising results and is suitable for further studies of the inflammatory process.     

An intramammary implant in the prevention of infectious mastitis in cows

A sterile polyethylene device was introduced in the milk cistern of two mammary quarters of forty dairy cows. The cows were divided into three groups according to the length of exposure of the device in the cistern (3 days, 14 days, 365 days). The somatic cell counts were studied for a year in the first 10 ml fraction of milk and an increase in the somatic cell counts was found in the quarters having the intramammary device, as compared with the control quarters (having no devices). This difference was marked during lactation and prior to the onset of drying off. For the reduction in the frequency of occurrence of new natural intramammary infections, the activity of the device against S. aureus was limited and no activity against S. agalactiae was proved. The proportions of polymorphonuclears, round-cell elements and macrophages were histologically studied and compared for the udder quarters with and without the intramammary body in correlation with the time of exposure of the device to the milk cistern milieu. The most marked differences in favour of the udder quarters with the intramammary device were recorded in the alveoli containing more cells with a significant proportion of polymorphonuclear leucocytes. Small differences were found in the interstitial and subepithelial zone of the milk cistern. The activity of acid and alkaline phosphatase and adenosine triphosphatase was histochemically determined in the tissue structures of the udder and was found not to change under the influence of the device. Leucocytes and macrophages adhering to the surface of the body were observed under scanning electron microscope.     

Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-alpha, beta1, and beta2

In contrast to other mastitis pathogens, the host response evoked during Staphylococcus aureus intramammary infection is marked by the absence of the induction of critical cytokines, including IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The elucidation of changes in the expression of other mediators with the potential to regulate mammary inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because these cytokines also have a demonstrated role in mediating inflammation, the objective of the current study was to determine whether S. aureus intramammary infection influences their expression. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk levels of TGF-alpha were evident within 32h of infection and persisted for 16h. Increases in TGF-beta1 and TGF-beta2 levels were detected within 40h of S. aureus infection and persisted through the end of the study. Thus, in contrast to IL-8 and TNF-alpha, S. aureus elicits host production of TGF-alpha, TGF-beta1, and TGF-beta2. This finding may suggest a role for these cytokines in mediating mammary gland host innate immune responses to S. aureus.     

The use of polyethylene intramammary device in protection of the lactating bovine udder against experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

The susceptibility of lactating bovine udder quarters fitted with a polyethylene intramammary device to infection was investigated. Following experimental challenge with Streptococcus agalactiae or Staphylococcus aureus, the incidence of infection was significantly (p less than 0.05) lower in intramammary device-fitted quarters compared to control quarters. In general, total foremilk and strippings milk somatic cell counts for intramammary device-fitted and control quarters were not significantly (p less than 0.05) different. Differential foremilk and strippings milk somatic cell counts were significantly (p less than 0.05) higher in samples from intramammary device-fitted quarters compared to control quarters.     

Specificity of the anti-inflammatory effect of a staphylococcal cell wall extract in the bovine udder

The cell wall of Staphylococcus aureus (strains 21 and Glaxo) was treated with deoxycholate and the insoluble residue was solubilised with lysozyme. The effect of the extract in modulating the inflammatory response due to infection of the lactating bovine udder was evaluated. Cows were infected with S. aureus strain 21 or Streptococcus agalactiae, with or without the cell wall extracts. The clinical response to infection was assessed, and milk samples collected up to 30 h were assayed for antitrypsin and NAGase levels, somatic cell count, and for the ability of whey to support bacterial growth. The extracts markedly reduced the inflammatory response elicited by both S. aureus and S. agalactiae, indicating the effect was non-specific. The extract from strain 21 was generally more effective than that from strain Glaxo.     

Prevalence and effects of intramammary infection in beef cows

Prevalence and effects of intramammary infection in 322 beef cows was determined during three calving intervals. Intramammary infection was confirmed in 37% of cows and 18.1% of quarters. Coagulase-positive staphylococci accounted for 17.9% of infections with Staphylococcus aureus isolated from 7.1% of cows. Coagulase-negative staphylococci and micrococci accounted for the remainder of infectious organisms. Butterfat and total protein levels were reduced 27.3 (P less than .05) and 25.5% (P less than .01), respectively, in milk from quarters infected with S. aureus. Somatic cell counts were elevated (P less than .001) with 3,827 X 10(3) cells/ml for S. aureus-infected quarters as compared with 555 X 10(3) cells/ml for uninfected quarters. Somatic cell counts were negatively correlated with 210-d calf weaning weights. Staphylococcus aureus-infected cows weaned calves weighing 19.1 kg less (P less than .01) than uninfected cows. At a present market value of $1.65/kg, economic losses were placed at $31.43/calf from cows infected with S. aureus in one or more quarters.     

Field trials on the prophylaxis of intramammary infections in pregnant heifers

The study was carried out in 5 farms on 174 pregnant heifers. Clinical examination of the udder and bacteriological tests of quarter secretion were performed between the 8th and 3rd week before parturition, and then the animals were divided into a control group (64 heifers) and 3 experimental groups and immediately treated. A group of 32 experimental heifers was injected once with antioxidants (Vitamin A--600,000 i.u.; Vitamin D3--200,000 i.u.; Vitamin E--1.5 mg/kg b.w., Selenium--0.022 mg/kg b.w., i.m.). The next group (26 heads) was intramammary infused with antibiotic DC product (cloxacillin). Heifers from last experimental group (52) were injected with lysosyme dimer in a single dose of 0.02 mg/kg b.w. Clinical and bacteriological examinations were made during the first week after calving. The presence of bacteria was found in secretion of 22.6-38.9% udder quarters in 56.2-71.2% of pregnant heifers. The number of infected quarters (cows) did not change distinctly in the first week after calving except the lysozyme dimer group, where a decrease by 30% was noted. The percentage of quarters with elevated somatic cell count was higher in antibiotic DC group and closely similar in the other groups. None of examined methods showed an acceptable prophylactic effect. Clinical mastitis cases during first week after parturition were mostly caused by Escherichia coli, Staph. chromogenes, Staph. simulans, Staph. aureus, Staph. hyicus, Str. uberis, Str. acidominimus and Enterococcus faecalis.     

Host-response patterns of intramammary infections in dairy cows

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.     

Removal of hair surrounding the teat and associated bacterial counts on teat skin surface, in milk, and intramammary infections

The effectiveness of monthly removal of hair surrounding teats on the reduction of teat skin surface bacteria, and the incidence of intramammary infection (IMI), was studied for 10 months in a dairy farm. A split udder design was used where hair was removed on one side, left or right, with the other side serving as a control. Controls and treatment sides were randomly applied in a systematic fashion to 218 cows. Standard milking time pre- and post-milking hygiene practices were applied to all udders during the trial. Collection of teat skin swab solutions preceded aseptic collection of milk samples, performed at monthly intervals, immediately prior to milking. Teat skin bacterial counts did not differ between control and treated teats. Incidences of IMI were similar for treatment when compared with control mammary quarters, as measured by total or by pathogen type. In a second study, the effect of hair removal on the bacterial content of milk was determined using 40 cows. Treatments and allocations were as described. Udder half milk, milk from both mammary quarters of each udder half, was combined and diverted into separate buckets. Buckets were thoroughly cleaned and sanitized between milkings. A portion of bucket milk was collected 24 h after removal of udder hair. The total milk bacterial counts, and counts of psychrotrophs and thermoduric organisms were not reduced by udder hair removal. Results do not suggest that removal of udder hair leads to an improvement in milk quality as determined by milk bacterial content in the herd studied.