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Article

Mastitis Pathogens Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis Selectively Alter TLR Gene Transcription in Sheep Mammary Epithelial Cells

by
Riccardo Tassi
,
Helen Todd
and
Keith T. Ballingall
*
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2024, 15(3), 1772-1783; https://doi.org/10.3390/microbiolres15030118
Submission received: 24 July 2024 / Revised: 29 August 2024 / Accepted: 30 August 2024 / Published: 4 September 2024
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Abstract

:
Despite the impact of mastitis on sheep production worldwide, the pathogenesis and host response to bacterial infection of the ovine mammary gland are poorly characterized. Studies in cattle highlight the significance of the mammary epithelium in pathogen recognition and the subsequent host response. The objective of this study was to assess bacterial adherence, invasion, and Toll like receptor (TLR) gene expression in primary sheep mammary epithelial cells (pMEC) following co-culture with the three principal mastitis pathogens of sheep, Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis. S. aureus was 140-fold more adherent than S. uberis and 850-fold more adherent than M. haemolytica. However, only S. aureus was internalized after 3 h of co-culture. TLR1, 2, 3, 4, 6, and 9 were shown to be constitutively transcribed by pMEC. M. haemolytica induced upregulation of transcription of TLR1, 2, 3, and 4. By contrast, S. uberis and S. aureus induced concentration-dependent transcription of TLR2 and TLR4 with a higher level of transcription in cells stimulated with the bacteria at a multiplicity of infection (MOI) of 200 compared to cells stimulated with a MOI of 20. These experiments define the range of TLR genes constitutively transcribed in sheep pMEC and show that bacterial infection has the capacity to regulate transcription in a species-specific and concentration-dependent manner.

1. Introduction

Mastitis, or inflammation of the mammary gland, represents one of most important diseases of production associated with sheep farming worldwide [1,2,3]. The most common cause of mastitis is an intramammary infection (IMI) by one of the bacterial pathogens, Mannheimia haemolytica, Staphylococcus aureus, or Streptococcus uberis [4,5].
Mastitis may present as a subclinical infection with limited impact on animal welfare or as a severe clinical disease, which is not only an animal welfare issue but can also lead to death or premature culling in dairy and meat sheep production systems [6]. IMIs impact milk quantity and quality [2], which influences production efficiency by restricting the rate of weight gain in suckling lambs [1,7]. Studies conducted mainly in cattle suggest that the nature of the response to an IMI is dependent on the early interaction between the invading bacteria and the host immune system [8,9,10]. Research has focused on the role of mammary epithelial cells (MEC) in shaping the early immune response to infection as they represent both a target and a first line of defense against invading pathogens (reviewed [11,12]). As differences in the range of pathogens associated with IMIs in cattle and sheep have been described [5,13,14], it is necessary to determine whether observations made in cattle also apply to bacterial species in the sheep host. In cattle, persistence of mastitis associated with E. coli or S. aureus infection is attributed to adherence and invasion of MEC [15,16], while variation in S. uberis adhesion to MEC was associated with virulence [17] and prevention of adhesion is a potential target for vaccination [18].
The early recognition and response to infection is mediated by a range of host receptors, including members of the Toll-like receptor (TLR) family. TLRs recognize and bind conserved pathogen molecules, termed pathogen associated molecular patterns (PAMP). For example, TLR2 recognizes lipoteichoic acid present in the cell wall of gram-positive bacteria [19]. TLR2 can also form heterodimers with TLR1 or TLR6, allowing recognition of bacterial triacylated and diacylated lipoproteins, respectively [20]. TLR4 recognizes lipopolysaccharides (LPS) present in the outer membrane of gram-negative bacteria, whereas TLR5 binds flagellin, a protein which constitutes a major component of bacterial flagellae [21]. While the above TLRs are located on the outer surface of the cell membrane, other TLRs, such as TLR3, 7, 8, and 9, recognize nucleic acids of viral or bacterial origin and have an intracellular distribution [22]. TLR ligand binding induces a signal-transduction cascade that results in the release of antimicrobial peptides, the production of pro-inflammatory cytokines TNF-α and IL-1β, and chemokines such as CXCL-8 (IL-8), which in turn initiates an inflammatory response and the recruitment of phagocytes and lymphocytes to the site of infection [19].
While the role of TLRs has been investigated in ruminant leukocytes [23] and dendritic cells [23,24], these receptors are also important in other cell types, including mammary epithelial cells [12]. The transcription of TLRs 1–10 in a range of ovine tissues has previously been described [25]. These include keratinocytes [25], corneal epithelium [26], and respiratory turbinate cells [27].
Studies conducted in cattle identified constitutive transcription of TLR2 and TLR4 in mammary tissues [28,29] and in cultures of primary MEC (pMEC). In addition, the strong systemic inflammatory response observed in mastitis following an E. coli infection has been linked with the production by MEC of pro-inflammatory cytokines following stimulation of TLR4 by LPS [30,31]. In contrast, although the lipoteichoic acid (LTA) of gram-positive S. aureus is recognized by the mammary epithelium through TLR2, this does not result in activation of the pro-inflammatory cascade, suggesting that the lack of an initiation of a local inflammatory response may contribute to persistence of gram-positive infections (reviewed by Wellnitz and Bruckmaier [32]).
Here, using pMEC isolated from the lactating mammary gland, we are able to investigate the early interactions between the mammary epithelium and sheep mastitis pathogens using an in vitro co-culture system.

2. Materials and Methods

2.1. Isolation and Preparation of Bacterial Strains

Three mastitis pathogen isolates were cultured from milk samples collected during a recent survey of a commercial Scottish sheep flock [5]. The isolates, M. haemolytica MRI T1-008, S. aureus MRI T1-020, and S. uberis MRI T1-034, were streaked from frozen stocks onto sheep blood agar plates (E&O Laboratories, Bonnybridge, UK) and cultured at 37 °C overnight to check morphology and purity. One colony was inoculated in 50 mL BHI and incubated at 37 °C for 16 h with shaking. The cultures were serially diluted 10-fold in PBS from neat to 10−9 cfu/mL, and 3 × 20 μL aliquots were plated on sheep blood agar plates to provide an accurate viable count. Bacterial suspensions diluted to the required concentrations were centrifuged at 4000× g for 20 min, and the pellets were resuspended in complete medium (DMEM (Gibco (Carlsbad, CA, USA) supplemented with 10% fetal calf serum, 4 mM glutamine, 1 μg/mL hydrocortisone (Sigma, Darmstadt, Germany), 0.5 μg/mL human recombinant epithelial cell growth factor (Sigma), and 0.5 μg/mL human recombinant insulin like growth factor-1 (Sigma) to a concentration of 1 × 109 cfu/mL.

2.2. Preparation of Sheep Primary Mammary Epithelial Cells (pMEC)

Sheep primary mammary epithelial cells were isolated from mammary gland tissue collected from a lactating Scottish Blackface ewe at postmortem. Mammary tissue was collected into a sterile 50 mL tube containing carrier medium (Hanks Balanced Salt Solution (HBSS), Merck Life Sciences, Glasgow, UK) supplemented with 100 μg/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL gentamicin, and 5 μg/mL fungizone [Sigma]). Approximately 5 g of tissue was minced into fine pieces and placed in 100 mL digestion medium (carrier medium containing 0.5 mg collagenase IV, 0.2 U DNase I, and 0.5 mg hyaluronidase per mL) and incubated under agitation at room temperature for 3 h. Cells were filtered sequentially through 1 mm and 100 μm metal strainers and centrifuged at 40× g for 5 min before re-suspension in 5 mL of complete medium and seeded in a T25 tissue flask for 20 min at 37 °C. The non-adherent cells were transferred to a new flask and allowed to settle overnight. Non-adherent cells were discarded, and the remaining cells were cultured with the addition of fresh complete medium. Residual fibroblast cells were eliminated from the culture by differential trypsinization. Briefly, cells were washed twice with PBS, and trypsin was added to the monolayer and removed as soon as the fibroblast cells were detached; epithelial cells were then trypsinized and transferred to new flasks. Growth medium was changed twice a week, and before cells reached confluence, cultures were passaged 1:2 and later up to 1:5 to provide bulk cultures for archiving in liquid nitrogen. On observation under an inverse microscope the cell population was composed of cobblestone epithelial-like cells and more elongated myoepithelial cells in an approximate proportion of 90:10.

2.3. Stimulation of pMEC with Each of the Three Major Mastitis Pathogens of Sheep

Primary sheep mammary epithelial cells were cultured in complete medium, trypsinized, and seeded at a concentration of 5 × 105 cells/well into six-well tissue culture plates. After 48 h, the supernatant was removed and the cells, approximately 90% confluent, were washed twice with PBS. Based on our earlier infection and attachment studies [33] and levels described in the literature [34], two mL of complete medium containing bacteria at 1 × 106 or 1 × 107 cfu/mL were added to provide a wide range of infection corresponding to MOI of 20 and 200, respectively. Each treatment was performed in duplicate and included untreated medium-only controls. After 3 h of co-culture at 37 °C, 5% CO2 the supernatant was removed, and cells were washed twice with 5 mL of warm PBS before the addition of 2 mL/well of RNA later solution (Sigma-Aldrich, St. Louis, MO, USA). Samples were archived at −80 °C until analyzed.

2.4. In Vitro Adhesion and Invasion Assays

Bacterial preparations were added to the supernatant of a confluent monolayer of pMEC in a six-well plate at a multiplicity of infection (MOI) of 100 (bacteria:epithelial cells). Cells were incubated for 3 h at 37 °C under 5% CO2. Culture supernatants were discarded, and each well was washed three times with warm PBS to remove non-adherent bacteria. Cells were lysed by adding 500 μL per well of 0.1% vol/vol Triton X-100 (Sigma-Aldrich) in PBS. Lysates were serially diluted in cold PBS, and 20 μL was plated in triplicate on blood agar plates (E&O Laboratories, Leicestershire, UK). Plates were incubated overnight at 37 °C, and colonies were counted. The concentration of bacteria (cfu/mL) associated with the cells (adhered to the surface and internalized) was calculated. In parallel, cells on separate plates were washed and incubated for 1 h at 37 °C in 5% CO2 with 1 mL per well of growth medium supplemented with 150 μg/mL gentamicin to kill extracellular bacteria [17]. Wells with supernatant alone were included to assess the viability of bacteria and the bactericidal efficacy of gentamicin. Cells were washed, lysed, plated, and counted as described above, and the concentration of bacteria (cfu/mL) internalized was calculated. The number of bacteria that adhered on the surface was obtained by subtracting the concentration of internalized bacteria from the total concentration of bacteria associated with the cells. The experiment was conducted three times, with the results based on the average of three wells in each replicate.

2.5. Extraction and Quality Control of RNA from Archived pMEC Samples

Total RNA was extracted from the archived samples using the RNA easy kit (Qiagen) following the manufacturer’s instructions. RNA was quantified using a nano-drop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and diluted to 2 ng/μL in nuclease-free water. The quality and integrity of RNA was assessed using a 2100 Bioanalyzer (Agilent technology, Santa Clara, CA, USA.), and an RNA integrity number [35] was calculated for each sample using the Bioanalyzer 2100 Expert software. Only samples with an RNA integrity number equal to or higher than 7.8 were used [35].

2.6. Quantitative Reverse Transcription PCR (RT-qPCR), for TLRs 1–10

RT-qPCR for TLRs 1–10 was performed using primers and probes previously described by Thonur et al. [24] using the Quanti Fast Probe Plus kit (Qiagen, Manchester, UK). The RT-qPCR reaction was set up in polypropylene 96-well PCR plates (Axygen, New York, NY, USA), with each well containing 6.25 μL Quantifast MMix, 2.25 μL nuclease-free water, 0.25 μL Quantifast RT Mix, 0.5 μL 50× ROX dye, 0.25 μL of 20 mmol probe, 1 μL of both 20 mmol forward and reverse primers, and 13 μL of RNA diluted to 2 ng/μL, in duplicate wells. For each plate, β-actin was included as the reference housekeeping gene [36]. Amplification was performed using an ABI 7500 thermocycler (Applied Biosystems, Waltham, MA, USA) under the following conditions: 50 °C for 2 min and 95 °C for 2 min for retro transcription followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s. The suitability of β-actin as the reference gene was confirmed by the low variability observed between the samples (standard deviation < 1).

2.7. Statistical Analysis

RT-qPCR data were analyzed using the 7500 software version 1.4.1 (Applied Biosystems). A cut-off value was set in the logarithmic phase of the amplification curves. Ct values were then determined for TLRs 1–10 and the β-actin reference gene. The ΔCt value of each sample was determined by subtracting the reference gene Ct from sample Ct. The results are presented as ΔCt for each treatment, and ΔCt values of each TLR were used as input for an analysis of variance (ANOVA) model using Genstat software (version 12, VSN International, Hemel Hempstead, UK). Bacterial species and concentration were used as treatment in the model. The interaction between these factors was also evaluated. Effects were declared statistically significant when p < 0.05. Post-hoc comparisons with least square difference (LSD) test were performed, and significant differences were declared at p < 0.05. Fold change (FC) variation of the transcription compared to the untreated sample was calculated with the 2−ΔΔCt method [37]. The reciprocal fold change of down-regulated genes was calculated as −1/FC.

3. Results

3.1. Adhesion and Invasion of Sheep Mammary Epithelial Cells

The capacity of the M. haemolytica, S. uberis, and S. aureus to adhere and invade sheep pMEC was tested. All three isolates adhered to pMEC after a 3 h incubation (Figure 1A). S. aureus (5.31 ± 0.4 Log10 cfu/mL) was shown to adhere approximately 140-fold more efficiently than S. uberis (3.17 ± 0.33 Log10 cfu/mL) and approximately 850-fold more efficiently than M. haemolytica (2.38 ± 1.01 Log10 cfu/mL) (p < 0.05). No significant difference in adherence was observed between M. haemolytica and S. uberis. Only S. aureus was shown to invade pMEC after a 3-h incubation (Figure 1B) and was recovered at a concentration of 4.2 ± 0.88 Log10 cfu/mL.

3.2. Quantitative Analysis of TLR Gene Transcription

Quantitative analysis of TLR transcription in pMEC identified constitutive transcription of TLR1, TLR2, TLR3, TLR4, TLR6, and TLR9. Transcription of TLR5, TLR7, TLR8, and TLR10 was not detected in untreated pMEC or in pMEC following bacterial co-culture. Changes to the level of TLR transcription following co-culture with M. haemolytica, S. uberis, and S. aureus is described below and summarized in Table 1 and Figure 2 and Figure 3.

3.3. TLR1

Co-culture of pMEC with M. haemolytica at both MOI 200 and 20 increased transcription of TLR1 by approximately 3.4-fold (ΔCt 12.31 ± 0.42 and 12.27 ± 0.43, respectively) compared to untreated cells (14 ± 1.2 ΔCt). Co-culture with S. aureus at MOI 200 increased transcription by 2.9-fold (ΔCt 12.60 ± 0.76), whereas no change in transcription was observed at MOI 20 (ΔCt 13.67 ± 0.52). No change in transcription was observed in cells co-cultured with S. uberis at both MOI 200 and 20 (ΔCt 13.50 ± 0.26 and 14.01 ± 0.54, respectively). Concentration of bacteria (i.e., MOI) had a significant effect only for S. aureus showing a 2-fold increase in transcription in cells stimulated with MOI 200 in comparison to MOI 20.

3.4. TLR2

Co-culture of pMEC with M. haemolytica increased transcription of TLR2 at both MOI 200 and 20 by approximately 32 and 21-fold (ΔCt 8.97 ± 0.1 and 9.58 ± 0.36), respectively, when compared to untreated cells (ΔCt 13.96 ± 0.40). Conversely, a decrease of 2.3 and 1.3-fold in transcription of TLR2 was observed in cells treated with S. uberis and S. aureus at MOI 200 (ΔCt 15.6 ± 1.37 and 14.94 ± 1.89, respectively). No change in transcription was observed following stimulation with these two pathogens at MOI 20 (ΔCt 14.09 ± 0.15 for S. uberis and 13.14 ± 0.19 for S. aureus). Bacterial concentration also had an effect as transcription of TLR2 increased for S. aureus by 3.5-fold and S. uberis by 2.8-fold in cells stimulated with MOI 200 compared to those stimulated with MOI 20.

3.5. TLR3

Co-culture of pMEC with M. haemolytica increased the transcription of TLR3 by approximately 1.5-fold at both MOI 200 (ΔCt 6.7 ± 0.24) and MOI 20 (ΔCt 6.78 ± 0.27), compared to untreated cells (ΔCt 7.31 ± 0.07). An increase in transcription of approximately 2-fold was observed following stimulation with S. aureus at MOI 20 (ΔCt 6.34 ± 0.42) but not at MOI 200 (ΔCt 7.07 ± 0.11), whereas no differences were observed in cells stimulated with S. uberis at both MOI 200 and 20 (ΔCt 7.20 ± 0.32 and 7.15 ± 0.1, respectively). Concentration of bacteria had a significant effect only for S. aureus with the transcription level approximately 2-fold higher in cells stimulated with MOI 20 than with MOI 200.

3.6. TLR4

Co-culture of pMEC with M. haemolytica increased transcription of TLR4 at both MOI 200 and MOI 20 by approximately 1.6 and 2.8-fold (ΔCt 7.56 ± 0.06 and 6.86 ± 0.53), respectively, compared to the untreated cells (ΔCt 8.26 ± 0.36). An increase in transcription of 1.9-fold was observed in cells stimulated with S. uberis at 200 MOI (ΔCt 7.39 ± 0.44). No differences were observed compared to the untreated cells after stimulation with S. uberis at 20 MOI (8.45 ± 0.69) or S. aureus at both MOI 200 and 20 (7.76 ± 0.27 and 8.69 ± 0.44, respectively). Bacterial concentration influenced all three species tested. The transcription level increased by 2.1-fold following stimulation with S. uberis and 1.9-fold for S. aureus at MOI 200 compared to MOI 20. The opposite trend was observed for M. haemolytica with the transcription level 1.6-fold higher in cells stimulated with MOI 20 than with MOI 200.

3.7. TLR6 and TLR9

Transcription of TLR6 and TLR9 genes showed no significant differences between treated and untreated cells (Table 1).

4. Discussion

Epithelial cells are one of the most abundant cell populations in a lactating mammary gland, and due to their anatomical location lining the lumen of the gland, they are likely to interact directly with ascending bacterial pathogens. Adhesion and entry into the mammary epithelium are virulence factors associated with pathogenesis, as intracellular bacteria are protected from some antibiotics, bacteria-specific antibodies, phagocytic cells, and from expulsion during lactation [12]. This allows the infection to invade deeper within the tissues and become persistent [15,38]. All three bacterial species tested in this study demonstrated the capacity to adhere to sheep pMEC. S. aureus bound with the highest efficiency and was the only bacterial pathogen internalized by the pMEC. This observation agrees with previous analyses of cattle MEC challenged with S aureus [16,39]. However, in contrast to our results, invasion of the mammary epithelium has been described for S. uberis in cattle [17,40] and M. haemolytica in sheep [41]. These differences in the capacity of S. uberis to invade may be due to subtle differences in the culture system or in the model itself as most of the cattle studies employed immortalized cell lines rather than the primary cell line used here. Differences in the capacity of M. haemolytica and S. uberis to invade may also be due to strain differences, which have been described for S. uberis [17], S. aureus [39], and E. coli [42].
During bacterial colonization of the mammary gland, a range of pattern-recognition receptors, including nucleotide-binding oligomerization domain-like receptors (NODs), RIG-like receptors, and TLR [43], located on the cellular membrane or within intracellular compartments of the mammary epithelial cells, are likely to be stimulated [11,12]. To extend the work of Thonur et al. [24] in which the TLR primers and probes are applied to ovine afferent lymph dendritic cells, this study also focused on expression of the TLR gene family but in mammary epithelial cells. We demonstrate that TLR2 is constitutively transcribed in sheep pMEC. The TLR2 receptor recognizes a range of bacterial ligands, including the lipoteichoic acid present in the cell wall of gram-positive bacteria such as S. aureus and S. uberis [44], and lipoproteins present in both gram-positive bacteria and gram-negative bacteria [45]. If we assume that TLR2 transcripts are translated into functional receptors, its presence indicates a role for TLR2 in the response of the mammary epithelium to these pathogens. Transcription of TLR2 was upregulated by co-culture with M. haemolytica, where it increased 32-fold compared with unstimulated cells. Such a substantial and rapid increase in transcription may result from the direct interaction between TLR2 and a bacterial PAMP such as one of the lipoproteins known to be expressed by M. haemolytica [46]. Alternatively, the effect may be indirect through interaction with other pattern-recognition receptors. Although no evidence of cross regulation of different TLR was found in cattle mammary epithelial cells [47], cross regulation has been observed in mouse intestinal mucosal epithelium, where in vitro stimulation with TLR5 ligands resulted in an upregulation of transcription of TLR2 and TLR4, whereas stimulation with TLR4 ligands resulted in a downregulation of TLR4 and an upregulation of TLR2 and the subsequent sensitization of the cells to TLR2 ligands [48].
TLR2 also forms heterodimers with TLR1 or TLR6, allowing recognition of bacterial triacylated and diacylated lipoproteins, respectively [20]. While both TLR1 and TLR6 are transcribed in the pMEC, TLR1 but not TLR6 increased approximately 3.4-fold in response to M. haemolytica, suggesting that regulation of TLR2 and TLR1 transcription may be linked and that recognition of triacylated lipoproteins may be important in the response to M. haemolytica infection.
Another well-characterized receptor is TLR4, which is involved in the recognition of lipopolysaccharides associated with gram-negative bacteria such as M. haemolytica in sheep and E. coli in cattle [47,49]. An increase in expression of TLR4 in mammary tissue may enhance the ability to respond to a gram-negative IMI. Here, we describe a 2.8-fold increase in TLR4 transcription following co-culture with M. haemolytica while transcription levels appeared to be dose-dependent following co-culture with the gram-positive bacteria S. uberis and S. aureus, which induced an increase in transcription at the highest MOI tested. In cattle, stimulation of TLR4 by E. coli-derived LPS is crucial for the host response. The early recognition of bacterial LPS enables a prompt host response consisting in the migration of polymorphonuclear leukocytes into the mammary gland [11], which reduces bacterial colonization. However, the response to LPS can itself cause clinical symptoms [47,50,51]. Similar to the response in cattle to E. coli mastitis, we predict that upregulation of TLR2 and TLR4 during an intermammary infection with M. haemolytica in sheep would amplify the host immune response, contributing to the acute pathology and severe symptoms that characterize this infection [52].
While TLR4 is not generally associated with gram-positive bacteria, an increase in TLR4 transcription in mammary glands of cattle infected with S. aureus has been described [28], although this was not associated with a strong inflammatory response [31]. As discussed for TLR2, it is possible that the increase in transcription observed here and, in the cattle studies, above is a consequence of the stimulation of other pathogen pattern-recognition receptors.
As described here, pMEC respond to an IMI by increasing TLR transcription within 3 h of contact with gram-negative pathogens. In contrast, the response to gram-positive pathogens appears to be more limited and dose-dependent. Transcription of TLR2 and TLR4 was not increased by co-culture with S. uberis and S. aureus at the lower MOI of 20, whereas an increase was observed at the higher MOI. This suggests that the lower number of bacteria in the initial stage of an infection would not cause an upregulation of these TLRs. The lack of substantial increases in TLR transcription may be a consequence of the failure of the mammary epithelium to recognize and respond to low concentrations of bacteria. This would facilitate the establishment of the intramammary infection due to the diminished or delayed host response [53,54].
In this study, TLR3 and TLR9 were also shown to be transcribed by the pMEC. These intracellular receptors recognize double-stranded viral and bacterial DNA, respectively. While TLR9 is considered important in the recognition of bacterial pathogens [55], transcription was not altered in this study, which supports earlier observations in cattle [28]. However, TLR9 was constitutively transcribed in pMEC and may play a role in S. aureus mastitis, as these pathogens are rapidly internalized by MEC, which may also limit the recognition by extracellular receptors such as TLR2. Transcription of TLR3 increased two-fold in response to S. aureus at MOI 20. The role of TLR3 in bacterial mastitis is unknown, as TLR3 recognizes molecules of viral origin; it is unlikely that S. aureus and S. uberis molecules are recognized by this receptor, and the increased transcription observed may be the result of cross regulation with other pattern-recognition receptors.

5. Conclusions

In conclusion, for the first time, we describe differences in the capacity of three major sheep mastitis-associated pathogens to adhere and invade primary mammary epithelial cells and the transcription of the sheep TLR gene family following co-culture. RT-qPCR data indicates that six sheep TLR genes, TLR1, 2, 3, 4, 6, and 9, are constitutively transcribed by pMEC in vitro. We observed significant changes in transcription of the TLR genes 1, 2, 3, and 4 after bacterial stimulation, which, if reflected in vivo, are likely to be important in directing the different responses to intramammary infection by gram-positive and gram-negative bacteria. Further studies to validate these data in vivo and at the protein level are planned. RNA-seq analysis will also provide a global analysis of changes in gene expression in sheep pMEC and in sheep mammary organoid cultures in response to co-culture with the range mastitis pathogens.

Author Contributions

Conceptualization, K.T.B. and R.T.; methodology, R.T. and H.T.; writing—original draft preparation, R.T. and K.T.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by program funding awarded to the Moredun Research Institute by the Scottish Government. For the purpose of open access, the author has applied a Creative Commons Attribution CC-BY license to any Author Accepted Manuscript version arising from this submission.

Institutional Review Board Statement

All tissue was derived from an animal at postmortem as part of a study which underwent local ethical approval, MRI ref: E09/17. (24 July 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are provided in this paper.

Acknowledgments

We would like to thank Biomathematics and Statistics Scotland (BioSS) for assistance with statistical analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

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Microbiolres 15 00118 g001
Figure 1. Adhesion to (A) and invasion of (B) of sheep primary mammary epithelial cells by M. haemolytica, S. uberis, and S. aureus. Results are expressed as the concentration of bacteria recovered, adhered (A), or internalized (B) after a 3 h incubation. The values represent the mean and the standard deviation of three replicates. No invasion was observed for M. haemolytica or S. uberis.
Figure 1. Adhesion to (A) and invasion of (B) of sheep primary mammary epithelial cells by M. haemolytica, S. uberis, and S. aureus. Results are expressed as the concentration of bacteria recovered, adhered (A), or internalized (B) after a 3 h incubation. The values represent the mean and the standard deviation of three replicates. No invasion was observed for M. haemolytica or S. uberis.
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Figure 2. Transcription of TLR genes following co-culture of pMEC with M. haemolytica (Man), S. uberis (Sub), and S. aureus (Sau) at MOI of 20 or 200. The untreated control provides constitutive transcription levels. For each treatment the average difference in CT is shown with the standard deviation. Significant differences in transcription compared to untreated cells are marked with an asterisk (*). For each species, significant differences (p < 0.05) in transcription associated with a different MOU are indicated with ‡.
Figure 2. Transcription of TLR genes following co-culture of pMEC with M. haemolytica (Man), S. uberis (Sub), and S. aureus (Sau) at MOI of 20 or 200. The untreated control provides constitutive transcription levels. For each treatment the average difference in CT is shown with the standard deviation. Significant differences in transcription compared to untreated cells are marked with an asterisk (*). For each species, significant differences (p < 0.05) in transcription associated with a different MOU are indicated with ‡.
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Figure 3. Relative transcription of TLR genes in pMEC co-cultured with M. haemolytica (Man), S. uberis (Sub), and S. aureus (Sau) at a multiplicity of infection of 200 and 20. Bars represent transcription and standard deviation relative to the untreated sample (Fold change).
Figure 3. Relative transcription of TLR genes in pMEC co-cultured with M. haemolytica (Man), S. uberis (Sub), and S. aureus (Sau) at a multiplicity of infection of 200 and 20. Bars represent transcription and standard deviation relative to the untreated sample (Fold change).
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Table 1. ANOVA analysis of TLR transcription data following co-culture with mastitis pathogens. For each TLR, bacterial species, bacterial concentration (MOI), and the interaction between species and concentration were considered.
Table 1. ANOVA analysis of TLR transcription data following co-culture with mastitis pathogens. For each TLR, bacterial species, bacterial concentration (MOI), and the interaction between species and concentration were considered.
TLRSpeciesConcentrationSpecies * Concentration
1<0.001 **0.1840.107
2<0.001 **0.015 *0.272
3<0.001 **0.0890.02
4<0.001 **0.086< 0.001 **
60.1470.1950.017
90.090.3260.274
* indicates significance at < 0.05 and ** indicate significance at < 0.01.
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MDPI and ACS Style

Tassi, R.; Todd, H.; Ballingall, K.T. Mastitis Pathogens Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis Selectively Alter TLR Gene Transcription in Sheep Mammary Epithelial Cells. Microbiol. Res. 2024, 15, 1772-1783. https://doi.org/10.3390/microbiolres15030118

AMA Style

Tassi R, Todd H, Ballingall KT. Mastitis Pathogens Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis Selectively Alter TLR Gene Transcription in Sheep Mammary Epithelial Cells. Microbiology Research. 2024; 15(3):1772-1783. https://doi.org/10.3390/microbiolres15030118

Chicago/Turabian Style

Tassi, Riccardo, Helen Todd, and Keith T. Ballingall. 2024. "Mastitis Pathogens Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis Selectively Alter TLR Gene Transcription in Sheep Mammary Epithelial Cells" Microbiology Research 15, no. 3: 1772-1783. https://doi.org/10.3390/microbiolres15030118

APA Style

Tassi, R., Todd, H., & Ballingall, K. T. (2024). Mastitis Pathogens Mannheimia haemolytica, Staphylococcus aureus, and Streptococcus uberis Selectively Alter TLR Gene Transcription in Sheep Mammary Epithelial Cells. Microbiology Research, 15(3), 1772-1783. https://doi.org/10.3390/microbiolres15030118

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