Relationship between the level of N-acetyl-beta-D-glucosaminidase (NAGase) in bovine milk and the presence of mastitis pathogens

Changes in the level of the tissue damage marker enzyme, N-acetyl-beta-D-glucosaminidase (NAGase) in quarter fore milks were found to be related to the presence and types of pathogenic bacteria present and to somatic cell counts (SCC). Minor pathogens (coagulase-negative staphylococci, Corynebacterium bovis) elicited a mild SCC increase (from a mean of 243 X 10(3)/ml in healthy quarters to 504 X 10(3)/ml in infected quarters) with marginal tissue damage (mean NAGase activity increased from 21 in healthy quarters to 28 in infected quarters). Major pathogens (i.e. Staphylococcus aureus, Streptococcus agalactiae, Str. dysgalactiae and Str. uberis) caused more severe tissue damage (mean NAGase of 48) and SCC increases (mean, 2803 X 10(3)/ml). The NAGase test could also be used effectively on composite milk samples where regular monthly NAGase analysis was able to identify correctly 74% of animals having infected quarters. The possibility of combining SCC and NAGase data in order to give a more definite diagnosis of bovine mastitis is discussed.     

Prediction of mastitis using milk somatic cell count, N-acetyl-beta-D-glucosaminidase, and lactose.

The objectives of this work were 1) to examine the responsiveness of SCC, lactose concentration, and NAGase activity in milk to changes in bacteriological status and 2) to develop models for predicting bacteriological status of mammary glands. Data included 550 cows in 10 commercial herds. Natural logarithm NAGase and log cell count were most responsive to changes in bacterial status. The log NAGase was relatively more effective in identifying major from minor pathogen infections, whereas log SCC was better able to differentiate between infected and uninfected classes. Non-transformed NAGase, SCC, and lactose were considerably less responsive to infection status. Logistic regression of bacterial status on herd, lactation number, milk, log SCC, log NAGase, and stage of lactation was performed. The least significant variables were removed in a stepwise process. Final predictors of infection status were herd, log SCC, and log NAGase. The role of log SCC was to discriminate infection from no infection, whereas log NAGase discriminated major from minor pathogens. The log NAGase, alone or in combination with log SCC, added substantially to the detection power of the model. Chi-square goodness of fit tests found no significant differences between observed and predicted infection probabilities. Substitution of herd averages for log SCC and log NAGase for the herd variables resulted in significant differences between predicted and observed herd infection probabilities.     

Effect of mastitis on proteolytic activity in bovine milk

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.     

N-acetyl-beta-D-glucosaminidase activity in whole milk and milk fractions

Experiment 1 was conducted to determine NAGase activity in skim, fat, and cell pellet fractions of foremilk and stripping milk from infection-free quarters. Changes in milk NAGase activity during a 12 h in vitro incubation were also determined. Eight cows, two quarters per cow, were used. One quarter of each cow received an intramammary infusion of oyster glycogen. N-Acetyl-beta-D-glucosaminidase activity was highest in stripping milk and in milk from infused quarters. The percentages of NAGase activity in skim, fat, and cell pellet fractions were 62.6, 22.4, and 12.6. The NAGase activity of milk incubated in vitro did not significantly change over time. Experiment 2 was conducted to determine if neutrophils lost NAGase activity during extravasation into milk. Leukocytosis was induced in infection-free quarters of five cows. The NAGase activities of peripheral neutrophils and milk neutrophils were not significantly different. Results from both studies suggest that the major source of milk NAGase is the mammary epithelial cell and that milk somatic cells contribute less than 15% of the total milk NAGase activity.     

Viability of milk neutrophils and severity of bovine coliform mastitis

To study the host-pathogen interactions during Escherichia coli mastitis, we first determined whether E. coli infection would change blood and milk polymorphonuclear neutrophil (PMN) chemiluminescence (CL) and viability. We then hypothesized that when E. coli invade the mammary gland, the viable PMN in milk would efficiently phagocytose and destroy E. coli before establishment of infection. We observed that the phagocytosis-dependent and independent CL were closely linked to PMN viability and were crucial to the outcome of mastitis. Maximal PMN influx and colony-forming units in infected quarters appeared at postinfection hours (PIH) 6 to 24. This further boosted PMN recruitment through bone marrow-blood barrier as well as blood-milk barrier. The survival of recruited PMN in the E. coli-infected quarters was much higher than that of noninfected quarters. Chemiluminescence activity of PMN from the infected quarters significantly increased following E. coli infection, even exceeding that of blood at PIH 6, 12, and 18 to 24; no such increase was observed in noninfected quarters, suggesting that the various responses of milk PMN to stimuli resulted largely from PMN viability. The highest CL intensity and durability was observed in milk PMN from infected quarters at PIH 12. Whereas an increased viability of PMN in the noninfected quarters was only significant at PIH 6, the viability of PMN in infected quarters was long lasting and significantly higher at PIH 6 to 72. Importantly, higher preinfection milk PMN viability correlated with bacterial clearance, which was accompanied by faster recovery. Our study strongly supports the hypothesis that boosting milk PMN viability could be a strategy with which to prevent or reduce the severity of coliform mastitis in dairy cows. This strategy might be achieved through strengthening bone marrow functionality.     

Interleukin-6 in quarter milk as a further prediction marker for bovine subclinical mastitis

Subclinical mastitis (SM) is a major concern in the dairy industry because it causes economic losses and can lead to clinical mastitis. The mechanisms of the onset and progression of SM are not fully understood, and a new procedure for the detection and appropriate prediction of SM leading to clinical mastitis is necessary for dairy cow management. Inflammatory cytokines such as interleukin (IL)-6 are known to be present in inflamed mammary glands at the onset of mastitis, and IL-6 concentrations correlate with the levels of inflammation. In this study, the detection of IL-6 was examined for the evaluation for the future prediction of SM in 77 quarter milk samples from 20 cows. IL-6 concentrations in quarter milk were measured by sandwich ELISA, and the data were compared with milk somatic cell count (SCC) levels to diagnose SM. Average IL-6 concentration was significantly higher in SM quarter milk (207·0 ± 441·6 pg/ml) than in healthy control quarter milk (12·6 ± 33·4 pg/ml, P<0·01). Results of the cross-tabulation table show that SM prediction accuracy based on IL-6 concentration was almost equal or superior to SM prediction accuracy based on SCC. The detection of IL-6 in milk indicated SM earlier than did the detection of elevated SCC. Thus, the detection of IL-6 in milk could be a future prediction marker for SM.     

Influence of bovine mastitis on lipolysis ond proteolysis in milk

Lipolysis and proteolysis in milk were determined before, during, and after experimentally induced mastitis. Streptococcus agalactiae was infused into one quarter of five cows to elicit an infection. Milk protease activity was higher during infection, but milk lipase activity was unchanged. Lipolytic damage to milk fat and proteolytic damage to milk casein occurred in the udder prior to milking during an infection. Lipolysis increased due to increased susceptibility of the milk fat to lipase action during infection. The mechanism of the increased susceptibility of the fat to lipolysis was not determined. After infections were eliminated, SCC, initial and stored FFA concentrations, and initial tryosine values returned to preinfection levels. However, after infections were eliminated, milk protease activity as determined by an increase in tryosine values remained elevated as milk SCC returned to preinfection levels. Protease activity returned to preinfection levels within 10 d after SCC returned to preinfection levels.     

Significance of the presence of bovine milk beta-glucuronidase in mastitis detection

The presence of beta-glucuronidase enzyme in bovine milk was related both to the existence of major and minor pathogens and to somatic cell counts. The detection of this enzyme in whole milk was made possible by the use of p-nitrophenyl-beta-glucuronide as a substrate. This detection allowed us to determine abnormal udder secretions with a high degree of specificity and sensitivity. The particular method of enzyme determination was considered important for mastitis detection because beta-D-glucuronidase, the most significant enzyme in inflammatory processes, is released selectively. The relationship between enzyme, presence of pathogens, and somatic cell counts was established in 220 milk samples obtained at random from individual quarters of apparently healthy udders of cows from four local dairy farms (Santiago del Estero and Tucuman, Argentina). Four of these samples were from cows of recent parturition and two from cows with severe clinical mastitis. Only 17% of the milk samples were normal with somatic cell counts 500,000 cells/ml or less. This ratio is the usual one throughout the area, and the remaining 83% showed higher somatic cell counts. Taking the latter as 100%, the presence of beta-glucuronidase and the positive bacteriological analyses represented 76 and 74%, respectively.     

Glycosidase activities in bovine milk over lactation

The presence and activity of glycosidases in Irish bovine milk over three months of lactation was investigated. A low level of variation between animals was observed and the highest level of glycosidase activity was present in colostrum, decreasing through transitional milk production to minimal but constant levels in mature milk. N-acetyl-β-d-glucosaminidase, α-l-fucosidase, α-galactosidase and N-acetyl-neuraminidase appear to be the most biologically relevant glycosidases in bovine milk. The elevated levels of enzymatic activity in colostrum suggests the milk glycosidases may play a role in the digestion of bovine milk glycans in the infant mammal, possibly acting as substitutes for bacterial glycosidases prior to colonisation by the gut microflora, which are involved in in vivo oligosaccharide metabolism in the lower gastrointestinal tract.     

A coagulase-negative variant of Staphylococcus aureus from bovine mastitis milk

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex? test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.     

Effect of subclinical mastitis on milk plasminogen and plasmin compared with that on sodium, antitrypsin and N-acetyl-beta-D-glucosaminidase

The effect of subclinical mastitis on levels of plasminogen and plasmin in milk from cows in a high-yielding herd was investigated. Comparisons were made with levels of milk Na, antitrypsin and N-acetyl-beta-D-glucosaminidase (NAGase). In samples from mastitic quarters plasminogen activity, as measured after activation to plasmin, increased by only 21% and plasmin by 82%, while NAGase increased by 307%. Plasminogen was the only component that was normally distributed, all other components showed more or less skewed distributions. Plasmin and plasminogen were significantly related to the other components. However, plasminogen plateaued when the other components continued to increase. There was thus no further increase in plasminogen with the severity of inflammation as with the other components. Plasmin showed a similar although less pronounced tendency. Results of treatment of mastitic whey samples with acid suggested that the non-linear increase in plasmin activity was due to interaction with acid-labile proteinase inhibitors. Mastitis led to dissociation of plasminogen and plasmin from the casein micelles. The degree of activation of plasminogen was higher with casein-associated than with soluble plasminogen in both healthy and mastitic milks. Plasmin was very closely related to milk Na, which is a sensitive indicator of epithelial integrity. It is suggested that plasmin contributes to Na leakage into milk by degrading membrane proteins of the epithelial lining. Plasminogen and antitrypsin, which are both plasma proteins, were not identically affected by stage of lactation, indicating nonidentical modes of transport from plasma to milk.     

Specific agglutination of Streptococcus agalactiae from bovine mastitis by casein components of bovine milk

Streptococcus agalactiae strains freshly isolated from bovine mastitis cases clumped within 15 min of addition of small amounts of bovine milk to a broth culture. This reaction was not observed with isolates from human infections or bovine strains that had been maintained in the laboratory for extensive periods. Intensity of the clumping response as measured by a microtiter dilution assay was highly variable. Analysis of several single colony isolates derived from one strain indicated that the clumping phenotype was genetically unstable. The clumping reaction took place in the presence of rifampicin or chloramphenicol. Milk components that caused aggregation were heat stable, relatively insensitive to proteases, and were larger than 10,000 daltons. Purified casein also induced clumping in these strains.     

Immunoproteomics to identify Staphylococcus aureus antigens expressed in bovine milk during mastitis

Staphylococcus aureus is an opportunistic pathogen affecting both human and animal species. An effective vaccine to prevent S. aureus bovine disease and transmission would have positive effects on animal well-being, food production, and human health. The objective of this study was to identify multiple antigens that are immunoreactive during udder colonization and disease for exploration as vaccine antigens to prevent bovine mastitis. Staphylococcus aureus produces several cell wall-anchored and surface-associated virulence factors that play key roles in the pathogenesis of mastitis. Many of these proteins are conserved between different strains of S. aureus and represent promising vaccine candidates. We used an immunoproteomics approach to identify antigenic proteins from the surface of S. aureus. The expression of cell wall and surface proteins from S. aureus was induced under low iron conditions, followed by trypsin extraction and separation by 2-dimensional electrophoresis. The separated proteins were blotted with antibodies from mastitic bovine milk and identified by liquid chromatography-mass spectrometry. Thirty-eight unique proteins were identified, of which 8 were predicted to be surface exposed and involved in S. aureus virulence. Two surface proteins, iron-regulated surface determinant protein C (IsdC) and ESAT-6 secretion system extracellular protein (EsxA), were cloned, expressed, and purified from Escherichia coli for confirmation of immune reactivity by ELISA. A PCR of 37 bovine S. aureus isolates indicated that the presence of esxA and isdC is conserved, and amino acid alignments revealed that IsdC and EsxA sequences are highly conserved. The immunoproteomics technique used in this study generated reproducible results and identified surface exposed and reactive antigens for further characterization.     

Hexose shunt dehydrogenase activity in leukocytes isolated from bovine milk

A spectrophotometric method for measuring small changes in light absorption at specific wavelengths from highly turbid suspensions of biological material (dual-beam spectrophotometry) has been used to measure glucose-6-phosphate oxidation and nicotinamide adenine dinucleotide phosphate (oxidized) reduction catalyzed by lysates of milk leukocytes. This reduction was correlated .84 with cellular protein concentration and, therefore, with cell concentration.     

Changes in N-acetyl-beta-D-glucosaminidase activities in relation to other milk components throughout normal lactation in the guinea pig

Changes in milk production, NAGase activities, Na, Cl, K, lactose, total N, and fat were followed throughout normal lactation in 26 healthy guinea pigs. Appropriate microassays were used and all determinations were performed on the same small skim milk sample (microhematocrit tube). A very sensitive and rapid spectrofluorimetric determination of NAGase in 10-microliters skim milk samples was evaluated. Four days after parturition, daily milk production reached a maximum of about 20 ml. Thereafter milk yield decreased progressively to about 7 ml on d 15. During the entire course of lactation, a progressive increase in NAGase, total N, fat, Na, and Cl was observed. However, K and lactose decreased. Highly significant positive correlations between NAGase and Na, Cl, fat, and total N were evident, whereas correlations were negative between NAGase and K, lactose, and milk production. These changes can be explained on basis of alterations that occur at the level of the blood-milk barrier. The short lactation period of the guinea pig, the easy handling and milking practices, together with the available sensitive micromethods can lead to the establishment of a convenient guinea pig mastitis model.     

Changes of physicochemical indicators during mastitis and the effects of milk ejection on their sensitivity

We examined the relationship between physicochemical indicators and somatic cells in the milk of dairy cows during experimentally induced mastitis and their significance as indicators for use in controlling udder health. We were concerned particularly with the effect of alveolar milk ejection on the sensitivity of these indicators. In Expt 1, Escherichia coli lipopolysaccharide (Esch. coli LPS) was injected into the left rear quarter to induce an inflammatory reaction in one quarter in each of six cows. The contralateral control quarter was injected with a solution of NaCl (9 g/l). Nine milk samples were taken from both quarters until 60 h after injection. In Expt 2, repeated milk samples were taken every 20 s from one quarter during a 120-s teat stimulation in 20 cows with different somatic cell counts (SCC). Quarters were clustered for low (<5.0 log cells/ml), mid (5.0-5.7 log cells/ml) and high (>5.7 log cells/ml) SCC of the sample taken at t=0 s. Samples were analysed for SCC, electrical conductivity (EC) and Na+ and Cl- concentrations. During the experimental inflammation SCC, EC, Na+ and Cl- peaked at 12 h from LPS administration and values in treated quarters (T) at this time were elevated to 7900, 157, 501 and 169% of the values in untreated quarters, respectively. In Expt 2, SCC, EC, Na+ and Cl- in high SCC quarters were 2520, 121, 283 and 141% of low SCC quarters at the start of stimulation (t=0 s), respectively. Highly significant (P<0.001) differences in EC, Na+ and Cl- between high and low SCC quarters disappeared owing to the onset of alveolar milk ejection 100 s after the first contact with the teat. In conclusion, SCC in cows' milk provided the strongest amplitude in the case of an intramammary inflammation. EC, Na+ or Cl- were useful tools only if the measurements were performed in cisternal milk before the start of alveolar milk ejection.     

Transfer of dietary zinc and fat to milk--evaluation of milk fat quality, milk fat precursors, and mastitis indicators

The present study demonstrated that the zinc concentration in bovine milk and blood plasma is significantly affected by the intake of saturated fat supplements. Sixteen Holstein cows were used in a 4 × 4 Latin square design with 4 periods of 12 d, and 4 dietary treatments were conducted. A total mixed ration based on corn silage, grass-clover silages, and pelleted sugar beet pulp was used on all treatments. A high de novo milk fat diet was formulated by adding rapeseed meal and molasses in the total mixed ration [39 mg of Zn/kg of dry matter (DM)], and a low de novo diet by adding saturated fat, fat-rich rapeseed cake, and corn (34 mg of Zn/kg of DM). Dietary Zn levels were increased by addition of ZnO to 83 and 80 mg of Zn/kg of DM. Treatments did not affect daily DM intake, or yield of energy-corrected milk, milk fat, or milk protein. The high de novo diet significantly increased milk fat percentage and milk content of fatty acids with chain length from C6 to C16, and decreased content of C18 and C18:1. Treatments did not influence milk free fatty acids at 4°C at 0 or 28 h after milking. The average diameter of milk fat globules was significantly greater in milk from cows offered low de novo diets. Furthermore, the low de novo diet significantly increased the concentration of nonesterified fatty acids and d-β-hydroxybutyrate in blood plasma, the latter was also increased in milk. Treatments did not affect the enzyme activity of lactate dehydrogenase and N-acetyl-β-d-glucosaminidase in milk or the activity of isocitrate dehydrogenase and malate dehydrogenase in blood plasma. The low de novo diet significantly increased plasma Zn and milk Zn content, whereas dietary Zn level did not in itself influence these parameters. This indicates that the transfer of fat from diet to milk might facilitate transfer of Zn from diet to milk.     

Antimicrobial resistance and genetic characterization of coagulase-negative staphylococci from bovine mastitis milk samples in Korea

Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species—namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.     

DNA carryover in milk samples from routine milk recording used for PCR-based diagnosis of bovine Staphylococcus aureus mastitis

Real-time PCR techniques are increasingly used to detect udder pathogens from milk samples collected non-aseptically at routine milk recording. The objectives of this study were (1) to estimate the statistical associations between cycle threshold (Ct) values for Staphylococcus aureus in non-aseptically collected composite samples taken at routine milk recording from cows milked consecutively with the same milking unit and milk meter; and (2) to formulate practical and plausible guidelines for understanding the diagnostic implications of PCR testing for Staph. aureus intramammary infection at routine milk recording. The study included 4 herds with conventional milking parlors and repeatedly low Ct-values for Staph. aureus (representing a high DNA load) in bulk tank milk. Composite milk samples were collected from all cows at all milking units during routine milk recording using the Tru-Test electronic milk meter (Tru-Test Group, Auckland, New Zealand) and analyzed using the PathoProof PCR (Thermo Fisher Scientific, Vantaa, Finland) assay. Milking clock times were retrieved at each milk meter to establish the milking order of the cows at each unit. A multinomial logistic regression was applied to estimate the association between Ct-values from cows milked consecutively with the same milking unit and milk meter. The following groups were selected based on Ct-values: (1) 0–31.3, (2) 31.4–33.9, (3) 34.0–37, (4) 37.1–39.9, and (5) 40 (negative result). The association between groups from cows milked consecutively with the same milking unit and milk meter was statistically significant. Approximately 60% of cows were in Ct group 5 if the antecedent cow was also in Ct group 5, but only 20% of cows were in Ct group 5 if the antecedent cow was in Ct group 1. The probability of cows being in Ct group 1 was not markedly influenced by the group of the antecedent cow. Statistical relationships in the intermediate range gave a plausible indication of a dose-response relationship. Carryover of bacterial DNA via the milking unit and milk meter is very likely to affect PCR results for Staph. aureus. Therefore, information about milking order must be considered in mastitis control efforts. We suggest a practical interpretation of PCR results: cows with a Ct-value <32 can be labeled “very likely to be infected with Staph. aureus,” but cows with Ct-values of >37 and 32–37 can be labeled “very likely to be negative for Staph. aureus” and “uncertain Staph. aureus status,” respectively.     

Suitability of milk lactate dehydrogenase and serum albumin for pathogen-specific mastitis detection in automatic milking systems

In response to intramammary infection (IMI), blood-derived leukocytes are transferred into milk, which can be measured as an increase of somatic cell count (SCC). Additionally, pathogen-dependent IgG increases in milk following infection. The IgG transfer into milk is associated with the opening of the blood-milk barrier, which is much more pronounced during gram-negative than gram-positive IMI. Thus, milk IgG concentration may help to predict the pathogen type causing IMI. Likewise, lactate dehydrogenase (LDH) and serum albumin (SA) cross the blood-milk barrier with IgG if its integrity is reduced. Because exact IgG analysis is complicated and difficult to automate, LDH activity and SA concentration aid as markers to predict the IgG transfer into milk in automatic milking systems (AMS). This study was conducted to test the hypothesis that LDH and SA in milk correlate with the IgG transfer, and in combination with SCC these factors allow the differentiation between gram-positive and gram-negative IMI or even more precisely the infection-causing pathogen. Further, the expression of these parameters in foremilk before (BME) and after (AME) milk ejection was tested. In the AMS, quarter milk samples (n = 686) from 48 Holstein-Friesian cows were collected manually BME and AME, followed by an aseptic sample for bacteriological culture. Mixed models were used to (1) predict the concentration of IgG transmitted from blood into milk based on LDH and SA; (2) use principal component analysis to evaluate joint patterns of SCC (cells/mL), IgG (mg/mL), LDH (U/L), and SA (mg/mL) and use the principal component scores to compare gram-positive, gram-negative, and control IMI types and BME versus AME samples; and (3) predict gram-positive and gram-negative IMI by inclusion of combined SCC-LDH and SCC-SA as predictors in the model. Overall, the SA and LDH had similar ability to predict IgG transmission from blood into milk. Comparing the areas under the curve (AUC) of the receiver operator characteristic curves, the SCC-LDH versus SCC-SA had lower gram-positive (AUC = 0.984 vs. 0.986) but similar gram-negative (AUC = 0.995 vs. 0.998) IMI prediction ability. The SCC, IgG, LDH, and SA were greater in gram-negative than in gram-positive IMI (BME and AME) in early lactation. All measured factors had higher values in milk samples taken BME than AME. In conclusion, LDH and SA could be used as replacement markers to indicate the presence of IgG transfer from blood into milk; in combination with SCC, both SA and LDH are suitable for differentiating IMI type, and BME is better for mastitis detection in AMS.