Subclinical mastitis (SM) is a common and economically significant disease of dairy cows, causing increased somatic cell counts (SCC) and decreased quality and yield of milk (Wilson et al. Reference Wilson, Gonzalez and Das1997; Pitkala et al. Reference Pitkala, Haveri, Pyorala, Myllys and Honkanen-Buzalski2004; Halasa et al. Reference Halasa, Nielen, De Roos, Van Hoorne, de Jong, Lam, van Werven and Hogeveen2009). Approximately 70 to 80% of mastitis losses are due to subclinical mastitis (Reneau & Packard, Reference Reneau and Packard1991). Subclinical mastitis infections are not evident and can persist in the mammary tissue throughout lactation (Pilla et al. Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013).
Subclinical mastitis is most commonly diagnosed by microbial culture-based (MC) methods or SCC, which are both traditional and well-established tests for detection of subclinical mastitis (Oliver et al. Reference Oliver, González, Hogan, Jayarao, Owens, Oliver, González, Hogan, Jayarao and Owens2004; Hand et al. Reference Hand, Godkin and Kelton2012). Although SCC is a robust quantitative measurement, it does not differentiate cell types. Microbiological culture is based on collection of quarter milks aseptically for inoculation on culture medium and further testing for microorganism identification. The requirement for aseptic collection of milk samples for MC can be a disadvantage as the process is susceptible to contamination. Furthermore, traditional methods using MC can be labor-intensive and it may take up to 2–7 days to reach a diagnosis (Barreiro et al. Reference Barreiro, Ferreira, Sanvido, Kostrzewa, Maier, Wegemann, Bottcher, Eberlin and dos Santos2010).
The milk leukocyte differential (MLD) has been investigated for potential in diagnosis of mastitis (Dulin et al. Reference Dulin, Paape and Weinland1982; Kelly et al. Reference Kelly, Tiernan, O'Sullivan and Joyce2000; Pillai et al. Reference Pillai, Kunze, Sordillo and Jayarao2001; Dosogne et al. Reference Dosogne, Vangroenweghe, Mehrzad, Massart-Leen and Burvenich2003; Schwarz et al. Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011a, Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czernyb; Pilla et al. Reference Pilla, Schwarz, Konig and Piccinini2012, Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013). The MLD can detect changes in proportions of cell types in milk independently of the SCC, which could provide information about inflammatory processes in quarters otherwise considered healthy (Pilla et al. Reference Pilla, Schwarz, Konig and Piccinini2012). This information could be useful when control programs for milk pathogens are being applied (Pilla et al. Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013). The changes in cell ratio have been used for the identification of inflammatory processes in cows with low SCC, with the potential to differentiate milk from healthy quarters from those with early or late inflammation (Pilla et al. Reference Pilla, Schwarz, Konig and Piccinini2012). The MLD patterns of 6 out of 41 quarter milk samples with SCC values from ≥9000 to ≤46 000 cells/ml were described by Schwarz et al. (Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011a) and their results revealed early inflammatory reactions based on the predominance of polymorphonuclear neutrophils (PMNL) (56–75%).
The MLD has been tested as an option to identify cows affected by any inflammatory process of the mammary gland, with the best results being reported by using logarithmic PMNL:lymphocyte ratio as the variable (Pilla et al. Reference Pilla, Schwarz, Konig and Piccinini2012). However, there is still little knowledge about the MLD and its application under field conditions. Therefore, the aims of this study were to evaluate the use of MLD to (a) identify quarter milks that are culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis.
Materials and methods
This research was approved by the North Carolina (NC) State University Institutional Animal Care and Use Committee (Raleigh).
Animals and herds
All lactating cows in herds A (170 cows) and B (172 cows) included in the most recent monthly Dairy Herd Improvement Association (DHIA) test results were screened for inclusion. From these cows, all lactating cows in each herd with a composite SCC > 200 × 103/ml, and with no history of clinical mastitis within the preceding month, were considered eligible for the study. The 78 selected cows included Holstein (n = 52), Jersey (n = 19) and cross bred cows (n = 19) in various lactations (1 to 7) and stages of lactation. Cows selected from the two NC dairy herds were subjected to detailed analysis of udder health status based on MLD and MC of aseptically collected quarter foremilk samples.
Cows on farms A (geometric mean bulk tank SCC = 112·3 × 103 cells/ml) and B (geometric mean bulk tank SCC = 71·3 × 103 cells/ml) were housed in free-stall and pack barn facilities, respectively, and were milked twice a day in parlors. Both herds had consistent application of mastitis control programs based on the recommendations of the National Mastitis Council (NMC; http://www.nmconline.org). In both herds, cows were fed a total mixed ration composed of corn silage, grain concentrate, and minerals, with access to hay. Water was available ad libitum. The farms were conventional milk producers with average milk yields of 9,015 (farm A) and 11,788 (farm B) kg/year per cow, respectively.
Milk sampling
Milk samples were collected from all functional quarters of the 78 eligible cows on the 2 farms. A total of 10 of the 312 possible quarters were non-functional. This left 302 quarter foremilk samples which were collected for MC according to NMC guidelines (Oliver et al. Reference Oliver, González, Hogan, Jayarao, Owens, Oliver, González, Hogan, Jayarao and Owens2004). Before milking, teat ends were scrubbed with 70% isopropanol and the first three squirts of milk were discarded. Ten milliliters of milk per mammary quarter were collected aseptically. After the foremilk sampling for MC, milk from each quarter was collected into a quarter-based sampling chamber (Advanced Animal Diagnostics Company, AAD, Inc., Durham, NC) for MLD analysis. Quarter foremilk samples for MC were refrigerated (4–7 °C) until further analysis.
Microbiologic analysis
All microorganisms were isolated and categorized using procedures consistent with those recommended by the NMC (Oliver et al. Reference Oliver, González, Hogan, Jayarao, Owens, Oliver, González, Hogan, Jayarao and Owens2004). Milk samples were plated within 24 h of collection. Milk samples were mixed and 0·1 ml of milk was inoculated onto trypticase soy agar plates with 5% sheep blood (Becton, Dickinson and Co., Sparks, MD). Inverted plates were incubated aerobically at 36 °C for 48 h and results were observed every 24 h regarding colony characteristics (shape, size, number, and color), haemolytic ability (presence and type), and possible contamination. Isolates were Gram stained and catalase reaction determined. Specific microbiology procedures are given in Supplementary File S1.
Somatic cell count
Monthly milk SCC were recorded from DHIA analysis, using composite milk samples with preservative (United DHIA, Radford, VA).
Milk leukocyte differential
Milk leukocyte differentials were determined on fresh milk collected within 15 days after the most recent DHIA test day. The instrument (QScout MLD® test, Advanced Animal Diagnostics, Inc., Durham, NC) uses fluorescent microscopy technology to count and differentiate immune cells in milk and was initially validated, as described in Supplementary File S2. In addition to providing absolute values for each cell type (neutrophil, lymphocyte and macrophage), the total leukocyte count and percentage and total of each cell type were reported and also used in an index to produce a categorical quarter diagnosis of healthy vs. infected (see Supplementary File S2). Phagocyte counts were calculated as the sum of macrophages and neutrophils. Because of the wide variations found within the cell populations, we evaluated the ratio among phagocytic cell groups expressed as a logarithm of base 10 with the aim of identifying a marker that indicated whether the quarters were more likely to be healthy or infected. The results were expressed as Log10 [Neutrophils/Lymphocytes] (Log10 (N/L)) and Log10 [Phagocytes/Lymphocytes] (Log10 (P/L)), as described previously by Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012).
In the current study, samples were processed in research mode (See Supplementary File S2) to increase accuracy of calculated differentials. The mid-lactation index was selected with the manufacturer-recommended threshold set at 7. The reader (QScout Farm Lab, Advanced Animal Diagnostics, Inc., Durham, NC) has programmable threshold levels that may be selected by the user. By changing thresholds, a user can weight results towards higher sensitivity or higher specificity. In order to assess performance at various thresholds, settings were evaluated for the index range of 1–12 to allow sensitivity and specificity vs MC to be evaluated at each threshold setting.
Subclinical mastitis definition
Mammary quarters were considered to have an intramammary infection (IMI) when quarter milk samples showed isolation of significant bacterial colony numbers as described by Arruda et al. (Reference Arruda, Godden, Rapnicki, Gorden, Timms, Aly, Lehenbauer and Champagne2013), with slight modification. Since we plated 0·1 ml of milk, we considered presence of IMI as detection of any pathogen at any level, similar to that described by Dohoo et al. (Reference Dohoo, Andersen, Dingwell, Hand, Kelton, Leslie, Schukken and Godden2011).
Quarters selected from cows with SCC > 200 × 103 cells/ml were categorized at quarter level according the following criteria as previously described (DVG, 2002; Bansal et al. Reference Bansal, Hamann, Grabowskit and Singh2005): (a) healthy: culture-negative and total leukocyte count (TLC) ≤ 100 × 103 cells/ml; (b) latent subclinical mastitis (latent-SM): culture-positive and TLC ≤ 100 × 103 cells/ml; (c) nonspecific subclinical mastitis (nonspecific-SM): culture-negative and TLC > 100 × 103 cells/ml; and (d) specific subclinical mastitis (specific-SM): culture-positive and TLC > 100 × 103 cells/ml.
Experimental design and statistical analysis
Data are presented as means ± se. Associations between the MLD and MC status of the udder quarters were analyzed by applying linear mixed models with the SAS® program (version 9.3; SAS Institute Inc., Cary, NC, USA) after testing for residual normality and homogeneity of variance. We included data from all foremilk samples without contamination and with complete results for MLD and MC. The statistical model included the fixed effects of herd, cow, position of the udder quarter, milk yield, lactation number, parity number, breed and IMI. Statistical significance was defined at P-value <0·05. The statistical model used in analysis is given in Supplementary File S3.
The analysis was performed on Log10 transformation for SCC, absolute values for each cell type, the total leukocyte count and percentage and total of each cell type to provide normal distribution of the data. Data were anti-log10 transformed for presentation of the results and discussion. The MLD sensitivity (Se) and specificity (Sp) were determined comparing the MC as a standard methodology with the categorical quarter diagnosis from MLD technology (healthy vs infected) using on-line statistical software (MedCalc for Windows, version 16·8, MedCalc Statistical Software, 2016).
Results
Microbiologic analysis
A total of 302 quarter milk samples was aseptically collected from eligible quarters. There were 8 contaminated samples, leaving 294 quarter samples with usable culture results. Frequency of mastitis pathogen identification by MC of 294 quarter samples is given in Table 1. Overall, 130 quarters (44·2%) were classified as culture-positive and 164 (55·8%) were negative on culture. Minor pathogens (n = 50) accounted for 17·0% of total samples, being composed of CNS (n = 38) and Corynebacterium spp. (n = 12). Coagulase negative staphylococci (CNS) were the most commonly isolated mastitis-causing pathogen. Among the CNS group, Staphylococcus chromogenes was the most frequent, being found in 24/38 CNS isolates (8·2% of all samples). A variety of other CNS species was found in the remainder (Table 1).
Table 1. Frequency of mastitis pathogen identification by microbiological culture of mammary quarter foremilk samples (n = 294) from two herds in North Carolina
† Other coagulase-negative staphylococci for Farms A and B, respectively, included S. capitis (0 and 1), S. hominis (1 and 0), S. hyicus (2 and 1), S. lugdnensis (2 and 0), S. sciuri (1 and 0), S. xylosus (1 and 0), and other staphylococci (4 and 1)
‡ Streptococci isolated from Farms A and B, respectively, included Str. bovis (2 and 0), Str. dysgalactiae (4 and 0), Str. uberis (12 and 2), and Aerococcus viridans (1 and 0)
§ Enterococci isolated on farms A and B, respectively, included Ent. avium (1 and 0), Ent. durans (0 and 1), Ent. faecium (2 and 0), and other enterococci (2 and 0)
Major pathogens were identified in 68 quarters (23·1% of all samples: Table 1). Out of these, 37 quarters had isolation of contagious pathogens, all S. aureus (12·6%). There were 31 quarters with environmental pathogens (10·5%), primarily streptococci. A total of 12 quarters (4·1%) were identified as positive for other miscellaneous pathogens (Nocardia spp., yeast and Prototheca spp.).
Somatic cell count and milk leukocyte differential
Comparison of MLD results for quarters with variable mastitis definitions (healthy, latent-SM, non-specific-SM and specific-SM)
There were 102 mammary quarters classified as healthy (35%), 32 as latent-SM (11%), 59 as nonspecific-SM (20·3%) and 98 as specific-SM (33·7%: Table 2). Mammary quarters with specific-SM (772·5 × 103 cells/ml), nonspecific-SM (527·1 × 103 cells/ml) and latent-SM (40·6 × 103 cells/ml) had higher TLC than healthy quarters (25·1 × 103 cells/ml). The neutrophils% were greater in specific-SM cases (65·7%) than nonspecific-SM cases (55·2%), latent-SM cases (55·0%) and healthy quarters (49·4%). Therefore, healthy quarters had the lowest mean value of absolute number of neutrophils (12·3 × 103 cells/ml). Although mammary quarters with latent-SM, nonspecific-SM and specific-SM had higher TLC than healthy quarters, the macrophages% were lower in quarters with specific-SM (12·3%), nonspecific-SM (17·3%) and latent-SM (23·0%), when compared to healthy quarters (28·9%). The lymphocytes% and phagocytes% were similar among tested groups, but mammary quarters with specific-SM, nonspecific-SM and latent-SM had higher mean value of absolute number of lymphocytes and phagocytes than healthy quarters (Table 2). We evaluated the ratio among phagocytic cell groups expressed as a logarithm of base 10 aiming to identify cows more likely to be milk culture-positive according to our definition of mastitis. We found that the cell ratio Log10 (N/L) was higher in quarters with specific-SM (0·57), nonspecific-SM (0·50) and latent-SM (0·49) than healthy quarters (0·44). Using the cell ratio Log10 (N/L) would provide some differentiation of the quarters classified according to our mastitis definition. On the other hand, there was no difference of the cell ratio Log10 (P/L) between quarters with specific-SM (0·66), nonspecific-SM (0·66), latent-SM (0·67) and healthy quarters (0·67) (Table 2).
Table 2. Mean values for individual cell populations and combinations of cell populations from quarter milk samples considering the mastitis definition (n = 291)
aCells were presented as absolute number and in ratio
aData are presented as means on Log10 transformation ± se
aData are presented as means on antiLog10 transformation between parentheses
aDifferent letters within row were significantly different (P < 0·05)
† Geometric mean of somatic cell count at cow level from most recent DHIA test day prior to quarter sample collection
‡ Total Leukocyte Count and other measures on quarter basis
§ Phagocyte count were based on the sum of macrophages and neutrophils
¶ Log10 (N/L) = Log10 [Neutrophils/Lymphocytes]
‡‡ Log10 (P/L) = Log10 [Phagocytes/Lymphocytes]
No influence of quarter position, milk yield, parity and breed could be found on milk MLD results. However, we observed an effect of stage of lactation (DIM1 = 4 to 100, DIM2 = 101 to 200 and DIM3 = 201 to 431) on milk MLD results. The greater the DIM, the greater the macrophages% (P < 0·04) and the phagocytes% (P < 0·01), but lower the lymphocytes% (P < 0·01) (Fig. 1).
Fig. 1. Effect of stage of lactation (DIM1 = 4 to 100, DIM2 = 101 to 200 and DIM3 = 201 to 431) on milk macrophages%, lymphocytes% and phagocytes%.
Performance of categorical analysis on instrument readout (negative or healthy vs positive or infected)
A total of 294 quarter samples were submitted to the automated technology based upon readout results. Three quarter milk samples were reported as disabled by the MLD automated technology, leaving results available for a total of 291 quarters (Table 2). Out of 102 mammary quarters designated as healthy, 98 quarters (96·1%) were categorized as negative and 4 (3·9%) as positive by the automated technology based upon readout results. A total of 32 quarters were classified as latent-SM, with 26 quarters (81·3%) classified negative and 6 as a positive. There were 59 quarters designated as having nonspecific SM, and the automated technology categorized 9 quarters as negative (15·3%) and 50 (84·7) as positive. Ninety-eight mammary quarters with specific-SM cases were categorized as 2 negative (2%) and as 96 (98%) as positive.
As shown in Table 2, the MLD categorized 156 mammary quarters as positive (53·6%) and 135 as a negative (46·4%). Out of all quarters categorized as negative (n = 135) by the MLD, 79·3% had negative cultures (n = 107) and 20·7% had positive cultures (n = 28). On the other hand, out of all quarters considered positive (n = 156) by the automated technology, 65·4% had positive cultures (n = 102) and 34·6% had negative cultures (n = 54). When MC was considered the gold standard for mastitis diagnosis, the calculated diagnostic Se of the MLD was 65·4% (IC95% = 57·4 to 72·8%) and the Sp was 79·3% (IC95% = 71·4% to 85·7%). Using MC results as the ‘gold standard,’ Se and Sp of the categorical instrument readout results (healthy or infected) based upon cut-offs ranging from 1–12 are shown in Fig. 2. Sensitivity progressively increased from a minimum of 50·4% at a user setting of 1 to a maximum of 71·3% at a setting of 12 (Fig. 2). Specificity progressively decreased from a maximum of 86·7% at user setting 1 to 66·7% at setting 12 (Fig. 2).
Fig. 2. Sensitivity and specificity evaluated at different threshold setting of QScout® MLD.
Comparison of MLD results for quarters following categorization by mastitis pathogen groups (minor, environmental, contagious and miscellaneous)
A total of 161 healthy quarters (culture-negative) were selected and compared to 130 infected quarters (culture-positive) according to the pathogen category. The MC and MLD results from mammary quarters infected with minor pathogens (n = 50; 17%), environmental pathogens (n = 31; 10·5%), contagious pathogens (n = 37; 12·6%), and miscellaneous pathogens (n = 12; 4·1%) were compared to healthy quarters (Table 3).
Table 3. Mean values for individual cell populations and combinations of cell populations from quarter milk samples considering the category of pathogens subclinical mastitis-causing (n = 291)
aCells were presented as absolute number and in ratio
aData are presented as means on Log10 transformation ± SE
aData are presented as means on antiLog10 transformation between parentheses
aDifferent letters within row were significantly different (P < 0·05)
† Geometric mean of somatic cell count at cow level from most recent DHIA test day prior to quarter sample collection
‡ Total Leukocyte Count and other measures on quarter basis
§ Phagocyte count were based on the sum of macrophages and neutrophils
¶ Log10 (N/L) = Log10 [Neutrophils/Lymphocytes]
†† Log10 (P/L) = Log10 [Phagocytes/Lymphocytes]
Mammary quarters subclinically infected by miscellaneous (992·4 × 103 cells/ml) and contagious (973·6 × 103 cells/ml) pathogens had a similar TLC, but both group of pathogens had higher TLC than healthy quarters (76·3 × 103 cells/ml) and quarters infected by environmental (332·2 × 103 cells/ml) and minor pathogens (134·4 × 103 cells/ml). Mammary quarters subclinically positive with miscellaneous, contagious, environmental and minor pathogens had higher mean values of absolute number of neutrophils and neutrophils% than healthy quarters. The absolute number of macrophages was higher in all infected quarters as compared to healthy quarters, however, the % macrophages was higher in healthy quarters than quarters infected by any pathogen. This represented a proportional decrease of macrophages% but increase of neutrophils% when a quarter became infected. The lymphocytes% and phagocytes% were similar among tested groups, but mammary quarters infected by any pathogen had higher mean numbers of lymphocytes and phagocytes as compared to healthy quarters (Table 3). The cell ratio Log10 (N/L) was significantly higher in quarters infected by miscellaneous (0·62), contagious (0·57), environmental (0·53) and minor pathogens (0·52) than in healthy contralateral quarters (0·47). On the other hand, there was no difference in the cell ratio Log10 (P/L) between healthy quarters (0·67) and quarters infected with miscellaneous (0·69), contagious (0·67), environmental (0·66) and minor pathogens (0·65) (Table 3).
Discussion
It has been proposed that the MLD can identify changes in relative cell populations before the increase in TLC occurs in the course of inflammatory process (Pilla et al., Reference Pilla, Schwarz, Konig and Piccinini2012, Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013). Based upon this, we asked if the use of MLD would be able to (a) identify quarter milks more likely to be culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis. We found that 65·4% of quarters producing MLD-positive test results were positive for MC, while 20·7% of quarters testing MLD-negative were culture-positive. The Log10 (N/L) ratios were shown to be the most useful ratio to differentiate specific subclinical mastitis cases from healthy quarters. In addition to giving a total cell count, the MLD can be used for more detailed evaluation of udder health status.
Microbiologic analysis
Both farms used for this study were representative of smaller farms with mastitis problems warranting investigation, in that a considerable number of various pathogens were detected including Staphylococcus aureus. The validity of using elevated composite cow SCC (>200 × 103 cells/ml in most recent test) as a criterion for selection was affirmed by the finding that an average of 44·2% of quarter samples tested produced a positive microbiological result (45·6% for farm A and 41·3% for farm B). Both farms had approximately the same profile of pathogens. We have found Staphylococcus aureus as a frequent problem in some dairies in our region. The CNS were frequently isolated, similar to other studies (e.g., Makovec & Ruegg (Reference Makovec and Ruegg2003); Tomazi et al. (Reference Tomazi, Goncalves, Barreiro, Arcari and Dos Santos2015)). Considering all isolates, there were 38·5% minor pathogens, with CNS predominating, 28·5% Staphylococcus aureus, 23·8% environmental pathogens with streptococci predominating, and 9·2% infrequent pathogens such as Nocardia spp., yeasts and Prototheca spp. The profile of pathogens found in positive cultures makes the herds used appropriate for an investigation of mastitis diagnostics considering multiple etiologies.
Somatic cell count and milk leukocyte differential
Comparison of MLD results for quarters with variable mastitis definition (healthy, latent-SM, non-specific-SM and specific-SM)
The significantly higher TLC for specific-SM (772·5 × 103 cells/ml) vs. healthy quarters (25·1 × 103 cells/ml) samples was not surprising, as it was part of the selection criteria. The magnitude of the difference, as well as the significantly higher total neutrophils, total macrophages, total lymphocytes, and total phagocytes was consistent with expectations, similar to other studies (Pillai et al. Reference Pillai, Kunze, Sordillo and Jayarao2001; Dosogne et al. Reference Dosogne, Vangroenweghe, Mehrzad, Massart-Leen and Burvenich2003; Schwarz et al. Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011a, Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czernyb; Pilla et al. Reference Pilla, Schwarz, Konig and Piccinini2012, Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013). Schwarz et al. (Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011a) showed that PMNL in milk samples with SCC values <6·25 × 103 cells/ml were rare (mean proportion = 15%). Pillai et al. (Reference Pillai, Kunze, Sordillo and Jayarao2001) evaluated the MLD from mammary quarters with high SCC (>250 × 103 cells/ml) in comparison to the quarters with low SCC (<250 × 103 cells/ml), and they observed that the TLC and PMNL were consistently higher in quarters with high SCC. Additionally, it was reported that quarters with high SCC, TLC and PMNL were more often positive on MC (62 to 87%) compared with those with low SCC, TLC and PMNL (37 to 51%).
Similar to our study, Pillai et al. (Reference Pillai, Kunze, Sordillo and Jayarao2001) observed that 33 to 49% (mean = 40%) of the inflammatory cells from infected quarters were PMNL, while PMNL constituted only 17 to 25% (mean = 20%) of the inflammatory cells counted from uninfected quarters. Schwarz et al. (Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011b) observed that PMNL were the dominant cell population in milk samples of diseased quarters, with proportions of PMNL ≥ 65%.
Results of our study are most comparable to those of Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012), who compared differential cell counts from 96 normal quarters with 92 abnormal quarters categorized as latent mastitis, unspecific mastitis and subclinical mastitis. Similar to our findings, Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012) found that lymphocytes, neutrophils, and Log10 (N/L) were significantly higher in abnormal quarters. Macrophages were not significantly affected in the study of Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012).
Our numerical results for neutrophils% were very similar to those reported by Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012). Although we detected differences in macrophages% between quarters with specific-SM vs. those with nonspecific-SM, latent-SM and healthy quarters, the absolute values we obtained for macrophages% were very similar to those of Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012). In general, in the present study there was a proportional decrease of macrophages% with increases of neutrophils% when the quarter became infected. This result was similar to those described by Schwarz et al. (Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011b), who reported a significant negative correlation between macrophage% and SCC.
In our study, in which we classified the mammary quarters in a slightly different manner, Log10 (N/L) mean values from healthy quarters (0·44) were significantly lower than the latent-SM (0·49), nonspecific-SM (0·50) and specific-SM (0·57) groups (Table 2). These mean values of Log10 (N/L) were lower than what Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012) reported. Pilla et al. (Reference Pilla, Schwarz, Konig and Piccinini2012) categorized quarters in four groups (healthy quarters, latent mastitis-LM, nonspecific mastitis-UM and subclinical mastitis-SM) according to the SCC and MC results. They found that the Log10 (PMNL/Lymphocytes) mean values in healthy quarters (0·11) were significantly lower than those in groups with latent mastitis (0·57), nonspecific mastitis (0·73), and subclinical mastitis (0·94). Similar to our study, Log10 (N/L) was significantly different in quarters with specific-SM (0·57) vs. healthy quarters (0·44), but not for Log10 (P/L), indicating the merit of investigating quarters with Log10 (N/L) > 0·44 because they may be more often infected. This value is similar to what Pilla et al. (Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013) reported as a cutoff value. This categorization of the quarters in different types of mastitis is important since it may minimize the effect of positive and false negatives, as an example the nonspecific-SM cases (even in absence of bacteria has high SCC).
According to Pilla et al. (Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013), no influence of sampling day, parity, lactation stage, or quarter position could be found on leukocyte differential results. We did not observe a similar finding based on our results, because the greater the DIM the greater the macrophages% and the phagocytes%, but lower the lymphocytes% which is in agreement with previous results (Dosogne et al. Reference Dosogne, Vangroenweghe, Mehrzad, Massart-Leen and Burvenich2003).
Performance of categorical analysis on readout (negative or healthy vs. positive or infected)
The MLD readout results corresponded reasonably well with the quarter culture results, with 79·3% of negative MLD results being negative on culture, while 65·4% of MLD-positive quarters were culture-positive. Our reported Se of 65·4% and Sp of 79·3% were similar to those reported in prior studies. Pilla et al. (Reference Pilla, Malvisi, Snel, Schwarz, Konig, Czerny and Piccinini2013) reported Se of 73·3% and Sp of 73·6%. Adjustment of user settings from 1 to 12 would allow user optimization of settings. Sensitivities progressively increased from 50·4% at setting 1 to 71·3% at setting 12, while specificities decreased from 86·7% at setting 1 to 66·7% at setting 12 (Fig. 2).
Comparison of MLD results for quarters following categorization by mastitis pathogen groups (minor, environmental, contagious and miscellaneous)
The difference in TLC was striking when quarters infected by any pathogen were compared to healthy quarters, as was the difference in neutrophils%, macrophages% and Log10 (N/L). Schwarz et al. (Reference Schwarz, Diesterbeck, Konig, Brugemann, Schlez, Zschock, Wolter and Czerny2011a) described results similar to our study, reporting significant differences of cellular components in milk between quarters infected with pathogens as compared to healthy quarters. Although we have observed differences of MLD between quarters infected by any pathogen vs. healthy quarters, we found that the MLD in response to SM cannot be used to specifically identify the causative pathogen.
One purpose of our study was to consider the actual field application of this technology. Milk culture or other forms of microbiological analysis can be costly to the producer. An obvious use of the MLD would be to focus on cows with monthly SCC above some cut-off point (here, >200 × 103 cells/ml) with screening the infection at quarter level by providing a more rapid diagnosis performed by automated technology based upon ‘on-farm differential cells’ readout results.
Two recent studies (Hockett et al, Reference Hockett, Payne and Rodriguez2014a, Reference Hockett, Payne and Rodriguezb) have evaluated the automated technology readout results for selective dry cow therapy after diagnosis of infection by MLD compared to blanket dry cow treatment with cephapirin benzathine and cloxacillin. These studies from Hockett et al. (Reference Hockett, Payne and Rodriguez2014a, Reference Hockett, Payne and Rodriguezb) indicated that the use of MLD to guide selective treatment of infected cows reduced the use of cephapirin benzathine (47%) and cloxacillin (58%), and resulted in similar rate of infection, SCC and milk compared to blanket antibiotic therapy.
Conclusion
The MLD response to subclinical mastitis can provide more detailed diagnostic evaluation of than provided by SCC alone. We found that 65·4% of quarters producing MLD-positive test results were positive for MC, with 20·7% of quarters testing MLD-negative found as culture-positive. Similar to other previous studies, quarters positive on culture had higher absolute numbers of neutrophils, lymphocytes and macrophages, with higher neutrophils% and lymphocytes% but lower macrophages%. The Log10 (N/L) ratios were shown to be the most useful ratio to differentiate specific subclinical mastitis cases from healthy quarters. An obvious use of the MLD would be to help focus on the cows with monthly SCC above some limit (here >200 × 103 cells/ml) for screening the infection at quarter level by providing a more rapid diagnosis performed by automated technology based upon ‘on-farm differential cells’ readout results. Although MLD was able to identify quarters more likely to be milk culture-positive it was not found useful to help identify the specific causative pathogen.
The authors are grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP) for scholarship (grant no. 2013/23613-8 and 2015/04570-1) and project funding (Proc. 2014/17411-6 and 2013/07914-8).
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029917000267
