Major Mammary Pathogens as Contributors to Total Bacterial Counts in Raw Milk

The purpose of this study was to assess the contribution of major mammary pathogens, as well as of coliform and Gram-negative non-coliform bacteria to standard plate counts (SPCs) of bulk tank milk samples (BTMSs). Randomly selected anonymous BTMSs were collected from 268 dairy herds (with approximately 29,000 cows) in the Czech Republic during 2007. The most frequently detected pathogens were found to be E. faecalis (16.1%; geometric mean 9.8 × 102 CFU/ml) and S. uberis (13.6%; 9.0 × 102 CFU/ml). Highly significant positive correlations (P < 0.01) between the bacterial counts of the dominant pathogens and the relevant SPC were found for E. faecalis and S. dysgalactiae, as were significant correlations (P < 0.05) for S. uberis, E. faecium, and S. aureus. Highly significant positive correlations (P < 0.01) were found between SPC and coliform count and between SPC and Gram-negative non-coliform bacteria count. The results suggest, therefore, a recent dominance of environmental pathogens especially streptococci and enterococci, over contagious mammary pathogens in BTMSs. The geometric means of SPC in BTMSs with mammary pathogen (7.7 × 104 CFU/ml for environmental; 7.4 × 104 CFU/ml for contagious pathogens) exceed significantly (P < 0.05) the geometric means of SPC of pathogen free BTMSs (4.4 × 104 CFU/ml). This study revealed that the major mammary pathogens contribute significantly to SPCs of BTMSs. Bulk tank milk samples, mammary pathogen count, standard plate count, coliform bacteria count, Gram-negative non-coliform bacteria count The standard plate count (SPC) of bulk tank milk samples (BTMSs) is the most widely accepted criterion for measuring milk quality in all milk-producing countries throughout the world. For example, both in Europe and the USA, SPC hygienic limit values are codified (Anonymous 2006; FDA 2001). Regulation No. 1662/2006 (Anonymous 2006) requires the SPC at 30 °C to be less than 1.0 × 105 CFU/ml for raw cows’ milk. In the USA, the Pasteurized Milk Ordinance (FDA 2001) states that, to qualify as grade A milk, bacterial counts shall not exceed 1.0 × 105 CFU/ml. Since early 1990s, researchers, progressive dairy producers, veterinarians and dairy health consultants have been interested in BTMSs analysis as a tool both to determine milk quality and to troubleshoot herds for mastitis (Jayarao et al. 2004). In general, microbial contamination of raw milk occurs from three main sources: from within the udder, from the exterior of the udder, and from the surface of milk handling and storage equipment (Bramley and McKinnon 1990; McKinnon et al. 1990; Jayarao and Wolfgang 2003). The health and hygiene of the cow are both important in influencing the level of microbial contamination of raw milk. Equally important are the temperature and length of time in storage (which allows microbial contaminants to multiply) and the types of bacteria present in bulk raw milk. Although there is often only one source of bacteria that causes high bulk tank counts, high bacteria counts can also result from a combination of factors (Murphy and Boor 2007). ACTA VET. BRNO 2009, 78: 455-461; doi:10.2754/avb200978030455 Address for correspondence: MVDr. Dušan Ryšánek, CSc. Veterinary Research Institute Hudcova 70 621 00 Brno, Czech Republic Phone: +420 533 331 501 Fax: +420 541 211 229 E-mail: rysanek@vri.cz http://www.vfu.cz/acta-vet/actavet.htm Foot-note: Staphylococcus aureus subsp. aureus in text only S. aureus Streptococcus dysgalactiae subsp. dysgalactiaein text only S. dysgalactiae Mammary gland pathogens have been a focus of interest as important contributors to the bacterial count in BTMS for many years, having been studied by Bramley et al. (1984), Hayes et al. (2001), Jayarao et al. (2004), Zadoks et al. (2004), and Howard (2006). The mastitis organisms most often found to influence the total bulk milk bacteria count are Streptococcus spp., followed by coagulase-negative staphylococci (CNS) and coliforms (Bramley and McKinnon 1990; Hayes et al. 2001; Zadoks et al. 2004; Howard 2006). Mammary gland pathogens can be listed in groups: major or minor, contagious or environmental (Fox and Gay 1993; Harmon 1994). Of the major pathogens, Streptococcus uberis, Streptococcus bovis, enterococci, coliforms, Pseudomonas spp., and Arcanobacterium pyogenes are classified as environmental. Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, coagulase-positive staphylococci, Staphylococcus intermedius, and Mycoplasma bovis are classified as contagious. The majority of these pathogens are considered in this study. Although the total plate count was less than 5.0 × 104/ml in raw milk from 81.5% of suppliers in the Czech Republic during 2003 (Anonymous 2004), for example, the legal limit has been exceeded in some cases. Therefore, the aim of this study was to clarify the proportions and bacterial counts of major mastitis pathogens in BTMSs and their relationships to SPC values. The purpose was to judge the contributions of these pathogens and groups of coliform and Gram-negative non-coliform bacteria to SPCs in BTMSs in order to improve microbial quality of raw milk intended for dairy processing. Materials and Methods Experimental design During January to July and September to November 2007, BTMSs were collected from 268 dairy herds (with approximately 29,000 cows) on regular test days scheduled for raw milk hygienic quality determination. The anonymous dairy herds were randomly selected over a 3-week test-day interval with a limitation of 30 herds per test day. The plate counts for specific major mammary gland pathogens and the SPCs were determined. In addition, coliform bacteria counts (CCs) and Gram-negative non-coliform bacterial counts (NCs) were undertaken. Staphylococcus aureus, other coagulase-positive staphylococci (CoPS), S. agalactiae and S. dysgalactiae were grouped as contagious pathogens while S. uberis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium were grouped as environmental pathogens, according to categorization by Fox and Gay (1993) and by Harmon (1994). Correlations were calculated for the bacterial counts of the relevant pathogens or groups of bacteria against SPCs. Sampling procedure All BTMSs intended for checking the hygienic quality of raw milk were collected in accordance with the European Standard EN ISO 707:1997 (Anonymous 1997) using auto-samplers (AutoSampler, Foss Electric, Hillerød, Denmark). All samples were collected by specially trained technicians from the bulk tank milk of raw milk suppliers. Samples were transported without a preservative reagent to the laboratory in polystyrene boxes containing freezer packs and examined immediately upon arrival. The technical design of the polystyrene boxes prevents the samples from becoming frozen. Microbiological examination The cultivation procedure was performed as per Jayarao et al. (2004). The milk samples were thoroughly mixed by gently inverting the milk vial 20 to 25 times. One millilitre of milk was transferred to a sterile tube containing 9 ml of sterile phosphate buffered saline (Sigma, Saint Louis, Missouri, USA). The 10-fold diluted sample was then vortexed at high speed for 15 s, and 50 μl was plated onto both selective and non-selective media. Plate count agar (HIMEDIA, Mumbai, India) and incubation for 72 h at 30 °C were used for enumeration of the SPC. CC was determined as lactose-positive colonies on MacConkey’s agar no. 3 (MCA) (Oxoid, Hampshire, UK) whilst NC was determined as a count of lactose-negative colonies on MCA. Escherichia coli were also determined on MCA. Streptococci counts were estimated using modified Edward’s medium (Oxoid) supplemented with colistin sulphate and oxolinic acid (Sawant et al. 2002). Baird Parker agar (Oxoid) was used to determine the number of staphylococci. Plates for enumeration were incubated at 37 °C for 48 h. Cetrimide agar (Merck, Darmstadt, Germany) with an incubation period of 48 h at 35 oC was used for isolation of P. aeruginosa. 456

The standard plate count (SPC) of bulk tank milk samples (BTMSs) is the most widely accepted criterion for measuring milk quality in all milk-producing countries throughout the world.For example, both in Europe and the USA, SPC hygienic limit values are codified (Anonymous 2006;FDA 2001). Regulation No. 1662/2006(Anonymous 2006) requires the SPC at 30 °C to be less than 1.0 × 10 5 CFU/ml for raw cows' milk.In the USA, the Pasteurized Milk Ordinance (FDA 2001) states that, to qualify as grade A milk, bacterial counts shall not exceed 1.0 × 10 5 CFU/ml.Since early 1990s, researchers, progressive dairy producers, veterinarians and dairy health consultants have been interested in BTMSs analysis as a tool both to determine milk quality and to troubleshoot herds for mastitis (Jayarao et al. 2004).
In general, microbial contamination of raw milk occurs from three main sources: from within the udder, from the exterior of the udder, and from the surface of milk handling and storage equipment (Bramley and McKinnon 1990;McKinnon et al. 1990;Jayarao and Wolfgang 2003).The health and hygiene of the cow are both important in influencing the level of microbial contamination of raw milk.Equally important are the temperature and length of time in storage (which allows microbial contaminants to multiply) and the types of bacteria present in bulk raw milk.Although there is often only one source of bacteria that causes high bulk tank counts, high bacteria counts can also result from a combination of factors (Murphy and Boor 2007).
Mammary gland pathogens have been a focus of interest as important contributors to the bacterial count in BTMS for many years, having been studied by Bramley et al. (1984), Hayes et al. (2001), Jayarao et al. (2004), Zadoks et al. (2004), andHoward (2006).The mastitis organisms most often found to influence the total bulk milk bacteria count are Streptococcus spp., followed by coagulase-negative staphylococci (CNS) and coliforms (Bramley and McKinnon 1990;Hayes et al. 2001;Zadoks et al. 2004;Howard 2006).
Mammary gland pathogens can be listed in groups: major or minor, contagious or environmental (Fox and Gay 1993;Harmon 1994) Although the total plate count was less than 5.0 × 10 4 /ml in raw milk from 81.5% of suppliers in the Czech Republic during 2003 (Anonymous 2004), for example, the legal limit has been exceeded in some cases.Therefore, the aim of this study was to clarify the proportions and bacterial counts of major mastitis pathogens in BTMSs and their relationships to SPC values.The purpose was to judge the contributions of these pathogens and groups of coliform and Gram-negative non-coliform bacteria to SPCs in BTMSs in order to improve microbial quality of raw milk intended for dairy processing.

Experimental design
During January to July and September to November 2007, BTMSs were collected from 268 dairy herds (with approximately 29,000 cows) on regular test days scheduled for raw milk hygienic quality determination.
The anonymous dairy herds were randomly selected over a 3-week test-day interval with a limitation of 30 herds per test day.The plate counts for specific major mammary gland pathogens and the SPCs were determined.In addition, coliform bacteria counts (CCs) and Gram-negative non-coliform bacterial counts (NCs) were undertaken.Staphylococcus aureus, other coagulase-positive staphylococci (CoPS), S. agalactiae and S. dysgalactiae were grouped as contagious pathogens while S. uberis, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis and Enterococcus faecium were grouped as environmental pathogens, according to categorization by Fox and Gay (1993) and by Harmon (1994).
Correlations were calculated for the bacterial counts of the relevant pathogens or groups of bacteria against SPCs.

Sampling procedure
All BTMSs intended for checking the hygienic quality of raw milk were collected in accordance with the European Standard EN ISO 707:1997 (Anonymous 1997) using auto-samplers (AutoSampler, Foss Electric, Hillerød, Denmark).All samples were collected by specially trained technicians from the bulk tank milk of raw milk suppliers.Samples were transported without a preservative reagent to the laboratory in polystyrene boxes containing freezer packs and examined immediately upon arrival.The technical design of the polystyrene boxes prevents the samples from becoming frozen.

Microbiological examination
The cultivation procedure was performed as per Jayarao et al. (2004).The milk samples were thoroughly mixed by gently inverting the milk vial 20 to 25 times.One millilitre of milk was transferred to a sterile tube containing 9 ml of sterile phosphate buffered saline (Sigma, Saint Louis, Missouri, USA).The 10-fold diluted sample was then vortexed at high speed for 15 s, and 50 µl was plated onto both selective and non-selective media.
Plate count agar (HIMEDIA, Mumbai, India) and incubation for 72 h at 30 °C were used for enumeration of the SPC.CC was determined as lactose-positive colonies on MacConkey's agar no. 3 (MCA) (Oxoid, Hampshire, UK) whilst NC was determined as a count of lactose-negative colonies on MCA.Escherichia coli were also determined on MCA.Streptococci counts were estimated using modified Edward's medium (Oxoid) supplemented with colistin sulphate and oxolinic acid (Sawant et al. 2002).Baird Parker agar (Oxoid) was used to determine the number of staphylococci.Plates for enumeration were incubated at 37 °C for 48 h.Cetrimide agar (Merck, Darmstadt, Germany) with an incubation period of 48 h at 35 ºC was used for isolation of P. aeruginosa.
Colonies suggestive of S. aureus and CoPS from Baird Parker agar were randomly selected, streaked onto 5% sheep blood agar (BA) (Blood Agar Base No. 2, HIMEDIA, Mumbai, India) and incubated for 24 h at 37 °C.Colonies expressing both typical and atypical (without zones of lipolysis and proteolysis) appearances were taken into consideration.The isolates were examined for haemolysis, catalase production, and for free coagulase using the tube coagulase test (ITEST, Hradec Kralove, Czech Republic).Coagulase-positive strains were examined using the Voges-Proskauer test for production of acetoin (VPtest, PLIVA-Lachema, Brno, Czech Republic) and were finally identified using STAPHYtest (PLIVA-Lachema).
Colonies suggestive of S. agalactiae, S. uberis, S. dysgalactiae, E. faecalis and E. faecium on Edward's agar supplemented with colistin sulphate and oxolinic acid were randomly selected, streaked onto BA and then incubated for 24 h at 37 °C.All isolates were examined for haemolysis and catalase production, differentiated using the CAMP-Esculin Test (NMC 1999), and their growth capabilities assessed on agar containing 6.5% NaCl and on agar containing 4% oxgall (OX-BILE desiccated, OXOID) (equivalent to 40% bile).The isolates were classified in Lancefield groups using the latex agglutination test (ITEST STREPTO GROUP, ITEST).A test for detection of pyrrolidonyl arylamidase (PYRtest, PLIVA-Lachema) was conducted and the isolates were identified using the STREPTOtest (PLIVA-Lachema).
Colonies suggestive of P. aeruginosa on Cetrimide agar were subcultured onto BA and, after 24 h incubation at 37 ºC, they were examined for oxidase production, citrate utilization, and lactose, sucrose and dextrose fermentation, as well as for motility (see above for media).
Bacterial count of specific pathogen was determined from percentage of verified colonies analogically as described in European Standard EN ISO 6888-1, 1999.
We used specific bacterial pathogen counts to determine the dominant pathogens (i.e. the bacterial species with the highest count, where two or more bacterial species were detected), and these formed the basic units for this study.

Statistical analysis
In order to be able to work with normally distributed data, SPCs, CCs, NCs and the bacterial counts of specific pathogens were adjusted using a logarithmic transformation (log base 10).
All statistical analyses were performed using the GraphPrism 5 for Windows software package (GraphPad Software Inc., San Diego, CA, USA).
The significance of differences between SPCs from BTMSs that were mammary pathogen-free and BTMSs containing contagious or environmental pathogens was assessed using unpaired t-test.
The dependency between the bacterial counts of specific pathogens and SPCs, as well as between SPCs and CCs or NCs, was expressed using Pearson's correlation coefficient (r).The significance of the respective correlation coefficients was verified by testing the null hypothesis r = 0.

Results
Mammary pathogens were detected in 77.4% of BTMSs, and 22.6% of the samples were pathogen-free.Table 1 summarizes the proportions and bacteria counts of the major mammary pathogens detected in BTMSs.The total proportion of BTMSs contaminated by environmental pathogens dominated over samples that were pathogen-free or contaminated by contagious pathogens.The most frequently detected pathogens were E. faecalis and S. uberis, whilst CoPS, E. faecium and S. agalactiae occurred with the lowest frequency.Based on the bacteria counts, the highest count was detected for E. faecalis and followed by S. uberis.The results suggest, therefore, a recent dominance of environmental pathogens, especially streptococci and enterococci, over contagious mammary pathogens in BTMSs.The geometric means of these bacteria count did not, however, exceed 1.0 × 10 3 CFU/ml.
As shown in Table 2, significantly higher (P < 0.05) values of SPC were detected for the contagious and environmental pathogen categories than in the case of pathogen-free samples.
Highly significant positive correlations (P < 0.01) were determined between the bacterial counts of dominant pathogens and the relevant SPCs for E. faecalis and S. dysgalactiae, as well as significantly positive correlations (P < 0.05) for S. uberis, E. faecium and S. aureus (Table 3).
There were highly significant positive correlations (P < 0.01) between SPC and both CC and NC Gram-negative categories (Tables 4 and 5).It means in other words that increasing SPCs in BTMSs could be accompanied by high contamination with coliform bacteria and/or Gram-negative non-coliform bacteria.

Discussion
The purpose of this study was to assess the recent contribution of major mammary pathogens and  streptococci and enterococci such as E. faecalis and S. uberis, along with E. faecium and S. dysgalactiae.These results are to a great extent in accordance with findings by other authors.Bramley et al. (1984) noted that high counts of mastitis pathogens in herd bulk milk samples were detected for herds with high incidence of S. uberis mastitis.Hayes et al. (2001) found S. uberis to be the predominant organism in BTMSs with SPCs ranging from 1.4 × 10 4 to 6.0 × 10 5 CFU/ ml.Zadoks et al. (2004) found that streptococci, staphylococci and Gram-negative bacteria, respectively, accounted for 69%, 3%, and 3% of total bacterial count variability, whilst the most commonly identified streptococcal species were S. uberis, Aerococcus viridans, and S. agalactiae, detected in 81%, 50%, and 31% of 48 bulk tank samples, respectively.Howard (2006) noted counts of > 1.0 × 10 3 CFU/ml for total aesculin-positive streptococci, CNS, and coliforms in 48%, 51%, and 11% of BTMSs, respectively.Finally, Murphy and Boor (2007) noted that, although other mastitis pathogens have the potential to influence the bulk tank count, Streptococcus spp., and most notably S. agalactiae and S. uberis, are the mastitis organisms that most often influence the total bulk milk count.
The significant correlation between the S. aureus counts in BTMSs and SPCs in this study was somewhat surprising.S. aureus is not thought to be a frequent contributor to total bulk tank counts (Murphy and Boor 2007).Zadoks et al. (2004) found that staphylococci accounted for only 3% of total bacterial count variability.Howard (2006) also found that, unlike the counts of aesculin-positive streptococci, counts for S. aureus were rather low, ranging from zero to 1.0 × 10 3 CFU/ml.In our study, however, we determined a positive correlation between S. aureus counts and SPC, despite low S. aureus counts (geometric mean = 3.4 × 10 2 CFU/ml).
This study revealed counts (geometric means) of SPC of 6.8 × 10 4 /ml and of CC and NC of 151 and 155 CFU/ml in 201 (75%) and 209 (78%) of 268 samples, respectively.Jayarao et al. (2004) had observed a CC count of 70 CFU/ml and a count of 200 CFU/ ml for NC; concurrent SPCs ranged from 1.8 × 10 2 to 6.3 × 10 4 / ml.In another study from Pennsylvania (USA) between 2000and 2001, Jayarao et al. (2001) ) reported that 126 BTMSs displayed 56% SPC < 5 × 10 3 /ml, 19% at 5 × 10 3 to 1 × 10 4 /ml, and 25% > 1 × 10 4 /ml.In general, it is accepted that CC > 100 CFU/ml suggests poor hygienic practice (Jayarao and Wolfgang 2003).This study revealed a high positive correlation between SPC and CC, as well as between SPC and NC.This finding corresponds only partly with the findings presented by Jayarao et al. (2004), who found that BTMSs with means NC > 200 CFU/ml were 6 times more likely to have medium SPCs and 4 times more likely to have high SPCs as compared with BTMSs with NC < 200 CFU/ml.On the other hand, these authors have determined the lack of a relationship between CC and SPC.Hayes et al. (2001) stated, however, that E. coli in particular has been shown to elevate bacteria counts in BTMSs.
Finally, it must be mentioned that the detection of such pathogens does not necessarily indicate that they originated from cows with mastitis.Potential environmental mastitis pathogens can occur in milk as a result of such other contributing factors as dirty cows, poor cleaning of equipment, and/or inadequate cooling of raw milk.An accompanying elevated somatic cell count, however, can sometimes serve as supportive evidence that mastitis may, especially unapparent mastitis, have caused an increase in the bulk milk bacteria count (Ryšánek and Babák 2005;Ryšánek et al. 2007;Murphy and Boor 2007).
In conclusion, the results of this study reveal that the most important recent contributors to elevated SPCs in BTMSs in the Czech Republic are streptococci and enterococci, especially E. faecalis along with S. uberis and E. faecium.S. aureus appears to be an important contributor to SPC from among contagious mammary pathogens.Based on correlation analysis, the most important contributors were E. faecalis and S. dysgalactiae, followed by S. uberis, E. faecium, S. aureus and the CC and NC categories of Gramnegative bacteria.This suggests that the healthy mammary gland is of equal importance as suitable environmental hygienic conditions for high quality of raw milk intended for dairy processing.
a -100% = 279 (in 11 cases two pathogens showed the same CFU values) b -Arithmetic means for logarithmic transformed data c -Standard deviation for logarithmic transformed data d -Back-transformed (i.e.geometric) means . Of the major pathogens, Streptococcus uberis, Streptococcus bovis, enterococci, coliforms, Pseudomonas spp., and Arcanobacterium pyogenes are classified as environmental.Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, coagulase-positive staphylococci, Staphylococcus intermedius, and Mycoplasma bovis are classified as contagious.The majority of these pathogens are considered in this study.

Table 1 .
Proportions and bacterial counts of dominant mammary pathogens in bulk tank milk samples

Table 2 .
Descriptive statistics and significance of differences for standard plate counts of the Free, Contagious, and Environmental pathogen categories in bulk tank milk samples a -Arithmetic means for logarithmic transformed data (the presence of different capital letters indicates statistical significance, i.e. two-tailed P-value for unpaired t-test was < a -Two-tailed P-value for null hypothesis r = 0 ( * P < 0.05; ** P < 0.01)

Table 4 .
Descriptive statistics for standard plate count, coliform bacteria count and Gram-negative non-coliform bacteria count in bulk tank milk samples