Product quality can be compromised both by the presence of mastitis and also from antibiotic residuals. These veterinary cases make the milk unfit for human consumption (Alhussien and Dang, Reference Alhussien and Dang2020). Cobirka et al. (Reference Cobirka, Tancin and Slama2020) presented a review about the epidemiology and classification of mastitis, discussing three microorganisms as the major pathogens that cause mastitis: Escherichia coli, Staphylococcus aureus and streptococci. Intra-mammary infections lead to a high somatic cell count (SCC) in milk. Some relationships have been observed: if SCC increases, the percentage of neutrophils increases too (Alhussien and Dang, Reference Alhussien and Dang2020). CD4⁺ lymphocytes are necessary for the antigen-specific recruitment of neutrophils to mycobacterial infection, possible acting via chemotactic cytokines (Yang, Reference Yang2018). There are only a few studies about the potential relationship between SCC (total SCC, neutrophils and CD4⁺ cells) and mastitis (clinical or subclinical) caused by E. coli and S. aureus (Rainard et al., Reference Rainard, Foucras, Boichard and Rupp2018).

We have recently demonstrated the utility of an automated flourescent cell counter for the simultaneous determination of SCC and neutrophil percentage in bovine milk (Chengolova et al., Reference Chengolova, Ivanov and Grigorova2021). Now we extend that work to determine the relationship of pathogenic microorganisms (Staphylococcus aureus, Escherichia coli and Coliforms) in milk samples to the SCC, neutrophil and CD4⁺ cell percentage.

Results and discussion

Determination of bacteria and somatic cell count in milk

In this research 103 quarter cow milk samples were analysed. SCC, neutrophil and CD4+ cells were determined. Microbiological analyses were also made – total bacteria count (TBC), Staphylococcus aureus, Escherichia coli and Coliforms.

First of all, the cow milk samples were categorized in three groups: Healthy, Dirty and Mastitic (online Supplementary Table S2). The Healthy group showed low total SCC (from 130 000 to 420 000 cells/ml) negative tests for differential bacteria count (Staphylococcus aureus, Escherichia coli and Coliforms) and low TBC (29 000 colony forming units per ml – cfu/ml). The threshold of 400 000 cells/ml for the Healthy group was chosen because healthy cows should have an SCC below 200 000 cells/ml, and cows with counts over 400 000 cells/mL should be considered as having an intramammary infection (Cobirka et al., Reference Cobirka, Tancin and Slama2020). The Dirty group, on the other hand, had almost the same range of SCC but showed extremely high TBC (918 180 cfu/ml ± 25%) and positive tests for differential bacteria count. Obviously, the proportion of Coliforms in dirty milk samples was significant. That was probably due to poor cow hygiene. That group of milk samples was excluded from the subsequent somatic cell analyses.

The Mastitic group had very large differences in SCC (from 280 000 to 1 650 000 cells/ml) and bacteria were detected. Also, it was observed that the count of Staphylococcus spp. was the highest compared to the E. coli and Coliforms in the Mastitic group.

It should be noticed that there are significant differences between the infections caused by S. aureus and E. coli. Intra-mammary infection by E. coli is acute in nature and generally clears within a few days (Goldberg et al., Reference Goldberg, Pankey, Politis, Zavizion and Bramley1995). In contrast, infection by S. aureus is often less severe but results in a chronic infection (Newbould, Reference Newbould1970). The Coliform organisms (Escherichia coli, Klebsiella spp. and Enterobacter aerogenes) also cause mastitis. The endotoxins contained in the bacterial cell wall have a pathogenic effect (Hohmann et al., Reference Hohmann, Wente, Zhang and Krömker2020).

Influence of different pathogenic microorganisms on SCC, neutrophil and CD4⁺ cell count

At the first stage of the study, 12 of the samples were classified as ‘Dirty’ so they were excluded from analysis, which as a consequence was made with 91 samples. The Healthy group comprised 16 samples and the Mastitic group 75 samples (detailed in online Supplementary Table S2). It was of interest to study the influence of the different mastitis causing pathogens on the proportion of different cell types – SCC, neutrophil and CD4⁺ cell count (Table 1).

Table 1. Influence of Staphylococcus aureus, Escherichia coli and Coliforms on SCC, neutrophil and CD4⁺ cell count

SCC, somatic cell count; n, number of milk samples.

Total and differential cell counts in the milk samples were determined by flow cytometer and automatic cell counter Lactoscan SCC. Table 1 shows the results obtained by both methods and they are relatively similar with negligible differences, suggesting that the Lactoscan device could be used for counting of SCC, neutrophils and CD4⁺ cells in milk. It was, however, apparent that the results obtained by the flow cytometer were higher than those obtained by Lactoscan, especially for the samples with high total number of microorganisms. Probably that was due to the difference in principles of the both methods and flow cytometric technique counted not only somatic cells but microorganisms or clotted huge proteins. However, the results remain similar.

It was noticed that if SCC increases, the percentage of neutrophils also increases. In this research the neutrophil count varied significantly from 17% in uninfected to 80% in mastitic milk samples. It is known that neutrophils are a key factor in the cows' defence against intra-mammary infection (Putz et al., Reference Putz, Palmer, Ma, Casas, Reinhardt and Lippolis2020). The highest neutrophil count in our samples was associated with Staphylococcus aureus mastitic samples. Blagitz et al. (Reference Blagitz, Souza, Batista, Azevedo, Benites, Melville and Della Libera2015) reported that Streptococcus dysgalactiae infected udders also had higher SCC and neutrophils but the number of the CD4⁻ had been higher, therefore, the number of the CD4⁺ decreased. Our observations on CD4⁺ cell in the samples showed increase in their count in samples in Mastitic group. Other authors reported similar observation. Rasheed et al. (Reference Rasheed, Usman and Niaz2020) presented a review about the milk samples with mastitis, where the number of CD4⁺ cells was higher than CD4⁺ cells in healthy milk samples. Besides that, the authors reported that the number of CD4⁺ cells was higher than CD8⁺ T-cells in the milk samples with staphylococcal mastitis, whereas in cows with streptococcal mastitis, parallel increase in both CD4⁺ and CD8⁺ T-cells were observed. In our case in the samples with staphylococcal mastitis the count of CD4⁺ cells increased compared to healthy milk samples, but in other mastitic samples the CD4⁺ cell count was lower than healthy milk samples. Taylor et al. (Reference Taylor, Keefe, Dellinger, Nakamura, Cullor and Stott1997) described that the lower value of CD4⁺ cells might be caused by the suppressive CD8⁺ lymphocytes, which inhibit proliferation of CD4 lymphocytes. In our study, CD4⁺ cell count was higher in healthy milk samples (about 8%) compared to mastitic ones (about 3%). Lower number of CD4⁺ cells (from 1 to 4%) was determined in samples positive for Staphylococcus spp. but those samples had lower SCC (from 2.7 to 4.0 × 10⁵ cells/ml). Also, the number of CD4⁺ cells in Staphylococcus spp.-positive samples increased (to 4.8%) together with elevated SCC. That was not observed in the other mastitic samples.

In conclusion, the findings of the present study suggest that the Lactoscan fluorescence cytometer is a good alternative to the flow cytometer for counting SCC, neutrophils and CD4⁺ cells. It was shown that the number of neutrophils and CD4 lymphocytes in the milk are a more reliable sign for differentiation between early and late phases of infection than SCC or bacteriological examination. The proportion of different cell types has been shown to be influenced by the type of pathogens.