Engler 2022

Microbial Pathogenesis 172 (2022) 105789
Contents lists available at ScienceDirect
Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath
Differential immune response to two Staphylococcus aureus strains with

distinct adaptation genotypes after experimental intramammary infection
of dairy cows
Carolina Engler a, b, María S. Renna a, b, Camila Beccaria a, b, Paula Silvestrini a, b,
Silvana I. Pirola a, b, Elizabet A.L. Pereyra a, b, Celina Baravalle a, b, Cecilia M. Camussone d,
Stefan Monecke e, f, Luis F. Calvinho c, d, Bibiana E. Dallard a, b, *
a
Laboratorio de Biología Celular y Molecular Aplicada, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Argentina
b
Instituto de Ciencias Veterinarias del Litoral (ICIVET-Litoral), Universidad Nacional del Litoral - Consejo Nacional de Investigaciones Científicas y Tecnológicas, (UNL-
CONICET), Argentina
c
Cátedra de Enfermedades Infecciosas. Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Argentina
d
Instituto de Investigación de la Cadena Láctea (INTA-CONICET), Estación Experimental Agropecuaria Rafaela, Ruta 34 Km 227, Rafaela, Santa Fe, Argentina
e
Institute for Medical Microbiology and Hygiene, TU Dresden, Dresden, Germany
f
Alere Technologies GmbH, Jena, Germany
A R T I C L E I N F O A B S T R A C T
Keywords: The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation
Bovine mastitis genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and
Experimental intramammary infection induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and
Staphylococcus aureus
5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm
Immune response
production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group,
Persistent and nonpersistent strains
pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated
with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was
characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188.
Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route
with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with
strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged
with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi.
In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the
one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters
inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at
d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and
lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest
SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of
IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoc
ulated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated
with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with
strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher
total IgG and IgG1 response; while strain 5011 (P) generated a higher IgG2 response (even against the heterol
ogous strain). The present study demonstrated that S. aureus strains with different genotype and adaptability to
bovine MG influence the local host immune response and the course and severity of the infectious process.
* Corresponding author. Bibiana E. Dallard. Instituto de Ciencias Veterinarias del Litoral (ICIVET-Litoral), R. P. Kreder 2805, (3080), Esperanza, Santa Fe,
Argentina.
E-mail address: bdallard@fcv.unl.edu.ar (B.E. Dallard).
https://doi.org/10.1016/j.micpath.2022.105789
Received 20 May 2022; Received in revised form 13 September 2022; Accepted 14 September 2022
Available online 20 September 2022
0882-4010/© 2022 Elsevier Ltd. All rights reserved.
C. Engler et al. Microbial Pathogenesis 172 (2022) 105789
1. Introduction mouse mastitis model induced by two S. aureus strains isolated from
bovine IMI with different clinical manifestations (P and NP), phenotypic
Staphylococcus aureus is still one of the most prevalent pathogens and genotypic profile [20]. This study revealed that the host immune
associated with bovine intramammary infection (IMI) worldwide, response was different for each S. aureus strain throughout the course of
causing important economic losses to dairy farming [1,2]. Early contact infection, showing in general a greater initial response to strain NP
of S. aureus with the bovine mammary gland is associated with a delayed compared with strain P and then a further immune response, mainly
and moderate inflammatory response compared with mastitis caused by stimulated by strain P and consistent with the development of a chronic
Gram negative bacteria (e.g. Escherichia coli) [3,4]. As a result these inflammatory process. Strain P, compared with strain NP, showed a
infections may very often establish and become persistent, leading not greater adaptation to the MG, inducing a higher immune response in the
only to increased transmission between cows during milking time, but advanced stages of IMI but with lower bacterial clearance from tissue
also to a loss of mammary function and fibrosis [5–7] which can in turn suggesting differential bacterial strategies for overcoming host immune
lead to culling of chronically affected cows. Classic antibiotic treatment response. In this model, considering that the animals used were similar,
in chronic cases is ineffective [8] and so far commercially available the immune response observed against strains bearing specific patho
vaccines have shown limited success to prevent S. aureus IMI [2]. genic traits implied that pathogen factors, rather than host factors, could
While significant advances have been made towards understanding influence the host response to achieve persistence in the MG [21].
the mechanisms employed by S. aureus to persist within the mammary Results from both previous studies carried out in vitro with MAC-T
gland (MG) [4], information available about host immune mechanisms cells and bovine macrophages [16] and in vivo in a mouse mastitis
evoked during chronic staphylococcal IMI is limited [9–12]. Chronic model [21] gave rise to the need to explore aspects of the immune
S. aureus IMI induces a sustained innate and adaptive immune response response induced by distinct S. aureus strains using an experimental
during lactation [9,10] or active involution [11,12] in bovine mammary challenge model for inducing bovine mastitis. A better understanding
tissue, however this response appears to be insufficient to eliminate the both of the pathogen and the immune response aspects is crucial to
pathogen. It has been suggested that several phenotypic and genotypic delineate alternatives to classic control practices and to refine current
characteristics are linked to S. aureus long-term persistence in the MG, strategies to intervene in the disease progress. Therefore, the aim of this
including the capacity to form biofilms and to invade cells and/or sur study was to evaluate and compare the ability of two S. aureus strains
vive intracellularly, the production of capsular polysaccharides (CP) and with different adaptation genotypes (low and high) to the bovine MG to
the accessory gene regulator (agr) type of the strain [13,14]. In a pre establish an IMI and induce an immune response after an experimental
vious study we used 20 selected S. aureus isolates from bovine IMI challenge in lactating cow.
categorized as persistent (P) and non-persistent (NP) based on clinical
behavior and presence of different genetic profiles. We demonstrated 2. Materials and methods
that S. aureus internalization into bovine mammary epithelial cells,
which is considered an early bacterial-host interaction, was 2.1. Cows
strain-dependent and that internalized bacteria overexpressed adher
ence and biofilm-forming genes, particularly those encoding FnBPs and Twelve clinically healthy Holstein dairy cows (4 cows/treatment
IcaD, compared with organisms that remained in coculture supernatants group) in mid lactation (weeks 31–36) from the School of Agriculture
[15]. and Livestock of Universidad Nacional del Litoral (UNL) were used.
Bacterial factors that contribute to intracellular persistence and host Cows were from parity 2 to 3, milked twice daily in a herringbone
factors leading to S. aureus clearance or survival during IMI are poorly milking parlor at 6:00 a.m. and 4:00 p.m., kept under grazing conditions
documented. To gain insights into the mechanism allowing S. aureus to (alfalfa pasture in the morning, ryegrass in the afternoon and corn silage
successfully persist intracellularly in the MG, in a recent study, we and alfalfa at night), received concentrates in the milking parlor (6.5 kg
evaluated and compared the ability of two S. aureus isolates from IMI per cow per day (18% CP) administered twice, 60% in the morning and
with different adaptation genotypes (low and high) to the bovine MG to 40% in the afternoon) and produced an average of 16 kg milk/day at the
adhere/internalize, persist, and induce damage in a bovine mammary beginning of the study. The cows had no previous history of clinical
epithelial cell line (MAC-T). In addition, we evaluated the phagocytic mastitis and had not received antibiotic or anti-inflammatory medica
and bactericidal capacity induced after the interaction between mam tion forty days before the initiation of the experiment. Selection of each
mary macrophages with both S. aureus strains [16]. These isolates group of cows was based on milk somatic cell counts (SCC) < 150 × 103/
harbored genes involved in adherence and biofilm production and ml, available from the local dairy herd improvement system, and
belonged to different CP type, agr group, pulsotype (PT) and sequence negative bacterial culture in all quarters. Quarter foremilk samples were
type/clonal complex (ST/CC) [16]. The non-persistent (NP) strain, aseptically collected (21, 14 and 7 days prior to challenge) and 48 h
selected for its low adaptation to bovine MG and characterized as agr before challenge according to standard procedures [21]. The first two
group II, cap5 positive, ST350 and weak biofilm producer, showed a low streams of milk from each teat were discarded, the next 5 ml were
adhesion/internalization and intracellular persistence capacity in collected in sterile plastic vials for bacteriological analysis and finally
MAC-T cells. Conversely, the persistent (P) strain, selected for its high ~20 ml were collected for SCC.
adaptation to bovine MG and characterized as agr group I, cap8 positive,
CC188 and strong biofilm producer showed high adhesion/internaliza 2.2. Bacterial strains and preparation of S. aureus inocula
tion and persistence capacity in MAC-T cells. Although the P strain was
recognized and phagocytized with greater efficiency by mammary Two S. aureus strains (designated 806 and 5011) isolated from milk
secretion macrophages compared with the NP strain, it showed greater samples from two Holstein cows with subclinical mastitis that belonged
resistance to microbicidal mechanisms. From these results we hypoth to two different dairy herds, were used for the intramammary challenge.
esized that the in vitro behavior of NP and P S. aureus strains regarding These strains were used in previous in vitro studies with MAC-T cells and
the ability to invade, persist and induce damage in MAC-T cells, could be bovine macrophages [16]. The S. aureus strain 806 was isolated only
associated with their in vivo behavior during a natural bovine IMI [16]. once from a mammary quarter of a cow in lactation and not re-isolated
Mastitis outcome is determined by pathogen virulence and cow’s in three consecutive milk samplings (7, 14 and 21 days) following
immune response [17]. According to several in vivo studies different standard treatment with a beta lactam antibiotic for 3 days. It was
S. aureus strains trigger differential innate immune responses which can considered to have low adaptation to the bovine mammary gland and
influence the course and severity of bovine mastitis [18,19]. In this re designated as NP. The S. aureus strain 5011 was isolated from the same
gard, in a previous study we investigated the immune response in a mammary quarter of a cow in consecutive monthly milk samplings over
2
a period of six months during lactation. It was considered highly adapted [23]: 0 = no overt changes in gland or milk, 1 = overt changes in milk
to the bovine MG and designated as P. To confirm that it was the same with no observed MG inflammation, 2 = local inflammation of the MG
strain of S. aureus that was isolated over a 6-month period, the genotypic accompanied by overt changes in milk and 3 = severe clinical mastitis
profiles of S. aureus isolates obtained from the same quarter were with systemic symptoms. Milk samples were taken from each inoculated
compared using pulse field gel electrophoresis (PFGE) [15]. Table 1 mammary quarter, pre-inoculation (time 0) and 0.5, 1, 2, 3, 4, 7, 14 and
shows a summary of phenotypic, genotypic and functional characteris 21 days pi. Following this experimental period, the mammary quarters
tics of S. aureus strains used in this study. inoculated with S. aureus strains 806 and 5011 were further evaluated
Before intramammary challenge, bacteria were activated from frozen by bacterial culture for 5 weeks at 7 day intervals (28, 35, 42, 49, and 56
stocks (− 80 ◦ C) by culture on Columbia agar base (CAB) (Britania, days pi) to monitor the development of chronic infections. General
Buenos Aires, Argentina) and incubated at 37 ◦ C for 24 h under aerobic condition and presence of clinical mastitis was daily evaluated during
conditions. Three colonies of each strain were inoculated into 5 ml of this period. After the whole observation period was over (day 56),
trypticase soy broth (TSB) (Britania) and incubated for 16 h at 37 ◦ C on a challenged animals were treated with intramammary antibiotics.
rotary shaker (150 rpm). Culture was vortexed and diluted 1:100 in TSB
(Britania) and incubated to mid-log phase for 2 h at 37 ◦ C on a rotary
shaker (150 rpm). Immediately before intramammary challenge, bac 2.5. Bacteriological examination, SCC and cell microscopic observation
teria were diluted to reach approximately 250 colony forming units
(CFU)/ml in pyrogen-free saline solution based on previous direct plate Milk samples were analyzed by standard bacteriological methods
counts carried out for each strain. and identity of challenge strains was confirmed by molecular analysis.
Milk samples (10 μl) were streaked onto blood agar plates supplemented
with 5% bovine blood and incubated for 48 h aerobically at 37 ◦ C. Plates
2.3. S. aureus intramammary challenge
were examined for bacterial growth at 24 h and 48 h. S. aureus was
presumptively identified based on the hemolytic pattern on blood agar,
A schematic overview of the S. aureus intra-mammary challenge
catalase and coagulase tests and differentiated from other coagulase-
model is given in Fig. 1. Of the 12 selected cows, 4 were inoculated in the
positive staphylococci by acetoin production and selective growth on
contralateral quarters (right front-RF and left hind-LH) with 1 ml of the
P agar with 7 μg/ml acriflavine [24]. The presence of one colony of
suspension containing S. aureus strain 806 (NP) (~250 CFU), 4 cows
S. aureus on blood agar was considered as a positive identification;
were inoculated in the contralateral quarters (RF and LH) with 1 ml of
therefore, detection limit was 100 CFU/ml. S. aureus colony counts
the suspension containing S. aureus strain 5011 (P) (~250 CFU) and 4
(CFU/ml) were performed from the challenged mammary quarters
cows were inoculated with 1 ml of pyrogen-free saline solution in the
immediately prior to infusion (d 0) and on days 0.5, 1, 2, 3, 4, 7, 14, 21,
contralateral quarters (RF and LH). We used a separate group of control
28, 35, 42, 49, and 56 pi. Identity of S. aureus isolates recovered
animals that were independent from any of the S. aureus challenged
following experimental challenge was confirmed by PFGE of SmaI-di
animals as we also aimed to evaluate the effect of the experimental
gested chromosomal DNA fragments using a CHEF-DR II apparatus
infection on the systemic immune response (manuscript in preparation).
(BioRad Laboratories, CA, USA) as described previously [25].
Intramammary inoculation of different bacterial suspensions and PBS
Quarter milk samples for SCC determination were preserved with
was performed after the afternoon milking as follows: teat ends were
azidiol (0.3%) at 4 ◦ C and analyzed within 24 h. The SCC was performed
thoroughly swabbed with 70% ethanol and then a sterile plastic cannula
by a commercial laboratory (Laboratorio Regional de Servicios Analí
attached to a disposable syringe was inserted through the teat canal.
ticos, Esperanza, Santa Fe, Argentina) using an automated counter
Following infusion, the teat was gently massaged in a dorsal direction
(Somacount 300, Bentley Instruments, Minesotta, USA).
[22]. Teats were dipped in 1% iodine teat dip after the infusion process.
In order to detect the presence of S. aureus inside milk cells, cytospins
An aliquot of the bacterial inocula and the pyrogen-free saline solution
from a pool of milk samples from mammary quarters (RF + LH) of each
were used for viable bacterial counts to determine the actual bacterial
animal were prepared. Only the cytospins of animals with positive
inocula and sterility check, respectively.
microbiological isolation in milk were stained. First, milk samples were
defatted by two centrifugations at 600×g 10 min. Pellet was resus
2.4. Progress of infection and sample collection pended in 1 ml of PBS and 100 μl of this solution was used per slide.
Cytospins were centrifuged at 30×g 10 min using a cytocentrifuge Cyto
The animals were monitored daily until 21 d post-inoculation (pi), Tek 2500 (Sakura). Then, were dried and frozen at − 20 ◦ C until staining
registering the general condition, MG clinical inflammation, appetite with May-Grünwald Giemsa Stain Kit (Abcam). Briefly, slides were fixed
and milk production. Local inflammatory changes of MG and milk were with methanol 2 min, stained for 3 min in May-Grünwald and by 1 min
detected following the scoring scheme proposed by Middleton et al. in distilled water. Then, the cytospin was incubated in Giemsa (one drop
Table 1
Summary of phenotypic, genotypic and functional characteristics of S. aureus strains used in this study. y Full characterization of strains in Ref. [16].
S. aureus Biofilm agr Capsule Adhesion Biofilm Penicillin Pulsotype Sequence Adherence/invasion and
strains† (MPA) type type genes producing resistance (PFGE) type (ST) persistence capacity#
genes *
Strain NP Weak agrII cap5 clfA, clfB, icaA, icaC, blaZ (− ) D ST350 Low
(806) fnbpA, icaD,
fnbpB (− ), fib, bap (− )
cna
Strain P Strong agrI cap8 clfA, clfB, icaA, icaC, blaZ O ST188 High
(5011) fnbpA, icaD, (CC188)
fnbpB (− ), fib, bap (− )
cna
References: NP: nonpersistent. P: persistent. MPA: microtiter plate assay. PFGE: pulse-field gel electrophoresis. (*) Evaluated by DNA microarrays. (#) Evaluated in a
bovine mammary epithelial cell line (MAC-T). Presence of capsular polysaccharaide genes 5 and 8 (cap5, cap8). Presence of clumping factor A and B genes (clfA, clfB);
fibronectin binding proteins A and B genes (fnbpA, fnbpB); fibrinogen binding protein gene (fib); collagen adhesion gene (cna). Presence of intercellular adhesion genes
A, C and D (icaA, icaC, icaD) and biofilm-associated protein gene (bap). Presence of beta-lactamase gene (blaZ).
3
Figure 1. Schematic overview of the S. aureus intra

mammary challenge model. Two of four quarters per
animal (right front-RF and left hind-LH) were inocu
lated with 1 ml of the suspension containing S. aureus
strain 806 (NP) (~400 CFU) or S. aureus strain 5011
(P) (~350 CFU) or with 1 ml of pyrogen-free saline
solution (controls). The animals were monitored daily
until 21 d post-inoculation (pi), registering the gen
eral condition, local inflammatory changes in MG and
milk, appetite and milk production. Milk samples
were taken from each inoculated mammary quarter,
pre-inoculation (time 0) and 0.5, 1, 2, 3, 4, 7, 14 and
21 days pi. Following this experimental period, the
mammary quarters inoculated with S. aureus strains
806 (NP) and 5011(P) were further evaluated by
bacterial culture for 5 weeks at 7 day intervals (28,
35, 42, 49, and 56 days pi).
per 1 ml of distilled water) for 15 min. Finally, slides were examined by 2.8. Cytokine immunoassay
light microscopy for the presence of stained cocci.
Samples were defatted following the same protocol as in Lf. To
2.6. Nitric oxide production determine levels of IL-1β, IL-6 and IL-4, commercial ELISA kits were
used following manufacturer’s instructions (Thermo Fisher Scientific,
Evaluation of NO production in milk was carried out by measuring its MA, USA). Briefly, the plate was sensitized overnight at RT with the
most stable metabolite, nitrite (NO2), following the methodology of capture antibody diluted 1/100 in carbonate/bicarbonate buffer 0.2 M,
Renna et al. [12] with modifications. First, milk samples were defatted pH 9.4 and subsequently blocked for 1 h at RT using D-PBS containing
by centrifugation at 1500×g 10 min. After removing the fat layer, 1 ml of BSA 4% and sucrose 5%. Then, samples were incubated in duplicate at
supernatant was treated with 1.5 ml of cold sodium acetate 0.1 M, pH RT for 1 h (IL-1β and IL-6) or 1 h 30 min (IL-4). To perform the quan
4.0 to precipitate caseins. Samples were clarified by centrifugation tification, a standard curve was constructed from known recombinant
(4000×g 10 min) and supernatants were incubated 10 min in a boiling bovine IL-1β, IL-6 and IL-4 concentrations provided in the kits. After
bath to inactivate proteases. After samples reached room temperature three washings with D-PBS Tween, samples were incubated with the
(RT), two more centrifugations were carried out at 10000×g for 10 and detection antibody diluted 1/100 for 1 h at RT. Subsequently, three
5 min. Once clarified supernatants were obtained, pH was adjusted to 7 washings were carried out and samples were incubated with
with sodium hydroxide 5 N. Processed samples were aliquoted and streptavidin-peroxidase (1/400) for 30 min at RT. The reaction was
stored at − 80 ◦ C until use. evidenced by incubation with TMB for 20 min and stopped with HCl 1 N.
This method allows for detection of nitrites formed by spontaneous Absorbance was read at 450 nm in a SPECTROstar Nano equipment.
oxidation of NO under physiological conditions [26]. Nitrite concen
tration was determined by Griess kit (Thermo Fisher Scientific, MA, 2.9. Analysis of immunoglobulins levels
USA). Briefly, 150 μl of clarified supernatants were placed in a 96-well
plate, 150 μl of Griess reagent was added and incubated for 30 min at Total IgG and IgG1 and IgG2 subtypes against S. aureus strains 806
RT in the dark. Finally, absorbance at 550 nm was measured using the (NP) and 5011 (P) were determined following the procedure described
SPECTROstar Nano equipment (BMG Labtech). Nitrite concentration by Renna et al. [12] with modifications. A 96-well microplate was
was calculated using a nitrite standard (sodium nitrite, provided in the sensitized with S. aureus lysates of these strains (10 μg/well) in bicar
kit). bonate buffer 0.1 M, pH 9 and incubated overnight at 4 ◦ C. S. aureus 806
(NP) and 5011 (P) strains lysates were obtained as described by
2.7. Lactoferrin concentration Camussone et al. [27]. After three washings performed with PBS-Tween
(0.05%) the plate was blocked for 1 h at 37 ◦ C with 5% of goat milk in
Milk samples were defatted by two centrifugations (1500×g, 10 min; PBS. Defatted milk samples were diluted 1/50 and incubated 1 h at
20800×g 30 min) at 4 ◦ C. Supernatants were aliquoted and frozen at 37 ◦ C. Then, three washings were carried out, and the plate was covered
− 80 ◦ C until use. Lf production was evaluated by a commercial ELISA kit with the detection antibodies: anti cow IgG-HRP (1/20.000) (Abcam),
(Bethyl Laboratories. Inc., Montgomery, USA). Briefly, microplate was anti cow IgG1-HRP (1/15.000) (Abcam), anti cow IgG2-HRP (1/1.000)
sensitized for 1 h at RT (20–25 ◦ C) with the capture antibody diluted 1/ (Bethyl Laboratories, Inc) and incubated 1 h at 37 ◦ C. Finally, after five
100 in carbonate/bicarbonate buffer 0.05 M, pH 9.6. After three washings TMB was added and the reaction was stopped after 5 min by
washings, the plate was covered with blocking solution (Tris-NaCl the addition of 1 N HCl. Absorbance at 450 nm was read in a SPEC
buffer, 0.05% Tween 20, pH 8) for 30 min at RT. Then, processed TROstar Nano equipment. Results were expressed as optical density
samples (diluted 1/1600) and reagents for constructing a standard curve (OD). In all cases for each immunoglobulin studied, plates were sensi
were placed in duplicate and incubated at RT for 1 h. After three tized with both S. aureus strains (806 and 5011) and cross reactivity
washings, samples were incubated with the peroxidase-conjugated effects between both strains were evaluated.
detection antibody (diluted 1/100000) for 1 h at RT. Reaction was
revealed with the incubation of chromogen 3,3′ , 5,5′ -tetrame 2.10. Statistical analysis
thylbenzidine (TMB) for 15 min and stopped with HCl 1 N. Absorbance
was read at 450 nm in SPECTROstar Nano equipment. Lf concentrations Statistical analysis of data was performed using SPSS 25.0 Software
for each sample were obtained by extrapolation from the standard curve (SPSS Inc., Chicago, IL). To determine the effect of IMI with different
made with recombinant bovine Lf provided in the kit. S. aureus strains on variables evaluated, data obtained were statistically
analyzed using repeated measures ANOVA (RMANOVA). ANOVA as
sumptions, such as normality of the distribution and homogeneity of
variances, were verified by the Kolmogorov-Smirnov and Levene tests,
4
respectively. One-way ANOVA was performed for each time evaluated, quarters challenged with S. aureus strain 806 (NP) received ~350 CFU
followed by Duncan’s multiple comparison test. Differences between while quarters challenged with the strain 5011 (P) received ~400 CFU/
time 0 (pre-i) and the rest of sampling times from quarters inoculated quarter. Similar numbers were recovered from the remainder of the
with both S. aureus strains were analyzed using one-way ANOVA fol S. aureus inocula that were used for the intramammary challenge. No
lowed by Duncan’s multiple comparison test. Differences of p < 0.05 bacterial growth was detected on pyrogen-free saline solution used for
were considered significant. Results were expressed as mean ± standard control quarters.
error of the mean (SEM).
3. Results 3.2. Bacteriological examination, SCC and cell microscopic observation
3.1. S. aureus intramammary challenge inocula Bacterial isolation and identification was carried out from milk
samples of quarters inoculated with S. aureus strains 806 (NP) and 5011
Based on viable bacterial counts of the prepared bacterial inocula, (P) and controls inoculated with saline solution at different times pi.
Results are detailed in Table 2.
Table 2
Isolations from mammary quarters experimentally inoculated with two selected S. aureus strains and mastitis score (0–1) at different times post inoculation.
Strain (animal – 0 0.5 1d 2d 3d 4d 7d 14 d 21 d 28 d 35 d 42 d 49 d 56 d
inoculated quarter) d
806 (NP) (Animal 1 (− ) (− ) 5000 1500 500 (− ) (− ) 100 300 (− ) (− ) (− ) (− ) (− )

RF) (0) (0) CFU/ml CFU/ml CFU/ (1) (1) CFU/ CFU/ (0) (0) (0) (0) (0)
(1) (1) ml ml ml
(1) (0) (0)
806 (NP) (Animal 1 (− ) (− ) 1500 500 500 (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
LH) (0) (0) CFU/ml CFU/ml CFU/ (1) (1) (0) (0) (0) (0) (0) (0) (0)
(1) (1) ml
(1)
806 (NP) (Animal 2 – – 300 (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) CFU/ml (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
(1)
806 (NP) (Animal 2 (− ) (¡) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
LH) (0) (0) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
806 (NP) (Animal 3 (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
806 (NP) (Animal 3 (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
LH) (0) (0) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
806 (NP) (Animal 4 (− ) (− ) 300 1500 (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) CFU/ml CFU/ml (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
(1) (1)
806 (NP) (Animal 4 (− ) (− ) (− ) (¡) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− ) (− )
LH) (0) (0) (1) (1) (1) (1) (1) (0) (0) (0) (0) (0) (0) (0)
5011 (P) (Animal 5 (− ) (− ) (− ) (− ) (− ) (− ) 250 380 (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) (0) (0) (0) (0) CFU/ CFU/ (0) (0) (0) (0) (0) (0)
ml ml
(0) (0)
5011 (P) (Animal 5 (− ) (− ) (− ) (− ) (− ) (− ) 200 320 (− ) 250 (− ) 100 (− ) (− )
LH) (0) (0) (0) (0) (0) (0) CFU/ CFU/ (0) CFU/ (0) CFU/ml (0) (0)
ml ml ml (0)
(0) (0) (0)
5011 (P) (Animal 6 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 400 (− ) 400 (− ) 250 100 (− )
RF) (0) (0) (0) (0) (0) (0) (0) CFU/ (0) CFU/ (0) CFU/ CFU/ (0)
ml ml ml ml
(0) (0) (0) (0)
5011 (P) (Animal 6 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 500 200 150 200 (− ) (− ) 150
LH) (0) (0) (0) (0) (0) (0) (0) CFU/ CFU/ CFU/ CFU/ (0) (0) CFU/
ml ml ml ml ml
(1) (0) (0) (0) (0)
5011 (P) (Animal 7 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 200 (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) (0) (0) (0) (0) (0) CFU/ (0) (0) (0) (0) (0) (0)
ml
(0)
5011 (P) (Animal 7 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 360 100 (− ) (− ) (− ) (− ) (− )
LH) (0) (0) (0) (0) (0) (0) (0) CFU/ CFU/ (0) (0) (0) (0) (0)
ml ml
(1) (0)
5011 (P) (Animal 8 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 340 (− ) (− ) (− ) (− ) (− ) (− )
RF) (0) (0) (0) (0) (0) (0) (0) CFU/ (0) (0) (0) (0) (0) (0)
ml
(0)
5011 (P) (Animal 8 (− ) (− ) (− ) (− ) (− ) (− ) (− ) 380 (− ) (− ) (− ) (− ) (− ) (− )
LH) (0) (0) (0) (0) (0) (0) (0) CFU/ (0) (0) (0) (0) (0) (0)
ml
(0)
RF: Right front quarter. LH: Left hind quarter. (− ) Negative microbiological culture to S. aureus.): Score (0) = no overt changes in gland or milk; Score (1) = overt
changes in milk with no observed MG inflammation (Middleton et al., 2004).
5
From quarters inoculated with strain 806 (NP) typical S. aureus in milk or apparent signs of inflammation (mastitis score 0) at any of the
yellowish colonies with double hemolysis halo were isolated at d 1, d 2 sampling times evaluated.
and at d 3 pi from RF quarter of animal 1, at d 1, d 2 and d 3 pi in LH SCC in milk samples from mammary quarters inoculated with both
quarter of animal 1 and only at d 1 pi in RF quarter of animal 2. Although S. aureus strains and control quarters inoculated with saline solution at
microbiological cultures were negative between days 4 and 7 in RF different sampling times was evaluated (Fig. 2). A significant effect of
quarter of animal 1, isolation was positive at 14 and 21 d pi and the S. aureus challenge was observed over time (p < 0.001), finding differ
morphology of colonies was similar to that observed in the first 3 d. ences in the SCC between the experimental groups. Regarding quarters
Complementary biochemical tests confirmed the presence of S. aureus in inoculated with strain 806 (NP), the highest SCC values were observed
the inoculated mammary quarters and the same original pulsotype was at d 2 pi, reaching a peak (mean) of 3.7 × 106 cells/ml. From d 7 pi there
recovered from these post-challenge samples (Fig. 1.; supplementary was a marked decrease that lasted until d 21 pi reaching basal values.
material). Quarters RF and LH of animal 3 did not yield a positive From mammary quarters inoculated with strain 5011 (P), no variations
microbiological result at any of the evaluated times. In RF quarter of were observed until d 3 pi; maximum values were reached at d 14 (3.8 ×
animal 4, isolation was positive at d 1 and d 2 pi and from LH quarter no 106 cells/ml) and remained high at 21 d pi. In control quarters, no
bacteriological growth was observed. Between 1 and 7 d pi, in quarters variations were observed during the sampling times studied (x 3.06 ×
with both positive and negative microbiological results, mastitis score 1 104 cells/ml).
was observed (i.e. small flakes and lumps without apparent inflamma
tory signs at MG palpation; Table 2). Cytospins from a pool of milk
samples from quarters (RF + LH) of animals 1 and 2 showed stained 3.3. Nitrites and lactoferrin production
cocci inside macrophages between days 1 and 3 pi (Fig. 2; supplemen
tary material). Nitrites and Lf production were evaluated in milk samples from
From mammary quarters inoculated with strain 5011 (P), typical mammary quarters inoculated with S. aureus strains 806 (NP) and 5011
S. aureus slightly yellowish colonies with double hemolysis halo were (P) and control quarters inoculated with saline solution at different
observed at 14 d pi in all quarters evaluated (average of 360 CFU/ml). In sampling times (Fig. 3A–B). A significant effect of S. aureus challenge
addition, bacterial growth was observed at 7 d pi in RF quarter and in LH was observed over time (p = 0.041), finding differences in nitrites
quarter of animal 5, and at 21 d pi in LH quarter of animals 6 and 7. The concentration between the experimental groups (Fig. 3A). Statistical
biochemical tests carried out confirmed the presence of S. aureus and the analysis showed a significant difference only at 21 d pi, when an in
same original pulsotype was recovered from these post-challenge sam crease in nitrite levels was found in mammary quarters inoculated with
ples (Fig. 1; supplementary material). Mastitis score 1 was only observed strain 5011 (P). Additionally, the S. aureus challenge effect in quarters
in milk of two LH quarters in animals 6 and 7 at 14 d pi, (i.e. small flakes inoculated with strain 806 (NP) was evaluated over time, showing an
and lumps without presence of local inflammatory signs; Table 2). increase in nitrite production at d 3 pi compared with time 0 (pre-i).
Cytospins from a pool of milk samples from quarters (RF + LH) of ani Control mammary quarters did not show variations throughout the
mals 5 and 6 showed stained cocci inside macrophages at 14 d pi (Fig. 2; experimental period (Fig. 3A).
supplementary material). A significant effect of S. aureus challenge was observed over time (p
For further monitoring the development of IMI, additional bacterial = 0.004) in Lf levels between the experimental groups (Fig. 3B).
cultures from quarters inoculated with S. aureus strains 806 (NP) and Maximum Lf concentrations were detected in mammary quarters inoc
5011 (P) were carried out for 5 weeks with 7 d intervals. Bacterial ulated with strain 806 (NP), showing an increase from d 3 pi. At d 21 pi,
growth was not detected in any of the samples taken from cows inocu Lf levels began to decline; however, they remained higher than those
lated with strain 806 (NP). Whilst from animals inoculated with strain observed in control quarters (p < 0.05). Although in quarters inoculated
5011 (P) S. aureus colonies were obtained from LH quarter (animal 5) at with strain 5011 (P) Lf levels were higher than in control mammary
28 d pi, in RF quarter (animal 6) at 28, 42 and 49 d pi and in LH quarter quarters, significant differences between groups were not detected at
(animal 6) at 28, 35 and 56 d pi; without macroscopic changes in milk or most of the evaluated times. At 4 and 21 d pi, in quarters inoculated with
apparent signs of inflammation (mastitis score 0). In every case identi strain 5011 (P) a significant increase of Lf levels was observed (p <
fication was carried out by standard biochemical tests and original 0.05); reaching at d 21 similar values to those found in milk from
pulsotype identity was confirmed. Microbiological analysis from control quarters inoculated with strain 806 (NP). Control mammary quarters did
mammary quarters did not show bacterial growth, macroscopic changes not show variations throughout the test (Fig. 3B).
Figure 2. Somatic cell count (SCC) in milk samples from mammary quarters inoculated with S. aureus strains 806 (NP) and 5011 (P) and controls inoculated with
saline solution at different times pi. The data are presented as means ± standard error of mean (SEM). Results of a repeated measure ANOVA (RMANOVA) are
indicated, different letters correspond to statistically significant differences (p < 0.05).
6
Fig. 3. A) Nitrite concentration (generated by spontaneous oxidation of NO). B)

Lactoferrin concentration. Assays performed in milk samples from mammary
quarters inoculated with S. aureus strains 806 (NP) and 5011 (P) and controls
inoculated with saline solution at different times pi. The data are presented as
means ± standard error of mean (SEM). Results of a repeated measure ANOVA
(RMANOVA) are indicated. Different letters correspond to statistically signifi
cant differences (p < 0.05). Asterisk represent significant difference between
time 0 (pre-i) and the rest of sampling times from quarters inoculated with 806
(NP) S. aureus strain (*p < 0.05).
3.4. Cytokine levels
IL-1β, IL-6 and IL-4 concentrations were evaluated in milk samples

from mammary quarters inoculated with S. aureus strains 806 (NP) and Fig. 4. Cytokines concentrations in milk samples from mammary quarters
5011 (P) and control quarters inoculated with saline solution at different inoculated with S. aureus strains 806 (NP) and 5011 (P) and controls quarters
inoculated with saline solution at different times pi. The concentrations of A) IL-
sampling times (Fig. 4A–C). A significant effect of S. aureus challenge
1β, B) IL-6 and C) IL-4 were all determined by ELISA. The data are presented as
was observed over time (p < 0.001) finding differences in the concen
means ± standard error of the mean (SEM). Results of a repeated measure
trations of IL-1β between experimental groups (Fig. 4A). In mammary ANOVA (RMANOVA) are indicated. Different letters correspond to statistically
quarters inoculated with strain 806 (NP) the maximum concentrations significant differences (p < 0.05).
of IL-1β were observed at d 2 pi and were higher than those observed in
control quarters and quarters inoculated with strain 5011 (P) (p < 0.05).
806 (NP) a significant increase was observed at d 1 pi (p < 0.05);
From d 3 pi a decrease was observed that lasted until d 21 pi; however,
however, the concentration decreased on d 2 reaching basal values. At
this cytokine concentration remained significantly higher (p < 0.05)
d 7 pi a second increase was observed and remained higher than control
during the observation period. In mammary quarters inoculated with
and quarters inoculated with strain 5011 (P) until d 21 pi (p < 0.05). In
strain 5011 (P), IL-1β concentrations were similar to those found in the
mammary quarters inoculated with strain 5011 (P) a significant increase
control quarters (Fig. 4A).
was observed at d 1 compared with control quarters (p < 0.05); how
A significant effect of S. aureus challenge was observed over time (p
ever, these values did not reach the IL-4 concentrations found in quarters
< 0.001) in the concentrations of IL-6 between the different experi
inoculated with strain 806 (NP). At the remaining sampling times
mental groups (Fig. 4B). In mammary quarters inoculated with strain
evaluated IL-4 concentrations were similar to those found in the control
806 (NP) a significant increase of IL-6 was observed from d 1 and
quarters (Fig. 4C).
continued until d 2 pi reaching the maximum concentrations (p < 0.05).
A marked decrease was observed on d 3 pi reaching basal values on d 4
pi. A second significant increase was observed on d 14 pi that lasted until 3.5. Levels of total and specific immunoglobulins
day 21 pi (p < 0.05). In mammary quarters inoculated with strain 5011
(P) IL-6 concentrations were similar to those found in the control The levels of total IgG, IgG1 and IgG2 were evaluated in milk samples
quarters (Fig. 4B). from mammary quarters inoculated with S. aureus strains 806 (NP) and
A significant effect of S. aureus challenge was observed over time (p 5011 (P) and control quarters inoculated with saline solution at different
< 0.001) in the concentrations of IL-4 between the different experi sampling times (Fig. 5A–F).
mental groups (Fig. 4C). In mammary quarters inoculated with strain In plates sensitized with strain 806 (NP), a significant effect of
7
Fig. 5. A) Total IgG, B) IgG1 and C) IgG2. Assays

performed in milk samples from mammary quarters
inoculated with S. aureus strains 806 (NP) and 5011
(P) and controls quarters inoculated with saline so
lution at different times pi. Plates were sensitized
with 806 (NP) strain. D) Total IgG, E) IgG1 and F)
IgG2. Assays performed in milk samples from mam
mary quarters inoculated with S. aureus strains 806
(NP) and 5011 (P) and controls quarters inoculated
with saline solution at different times pi. Plates were
sensitized with strain 5011 (P). Assays were all car
ried out by ELISA. The data are presented as means ±
standard error of the mean (SEM). Results of a
repeated measure ANOVA (RMANOVA) are indi
cated. Different letters correspond to statistically
significant differences (p < 0.05).
S. aureus challenge was observed over time finding differences (p = control quarters (p < 0.05). In plates sensitized with strain 5011 (P), the
0.030) in the total IgG levels between the experimental groups (Fig. 5A). IgG1 levels for both S. aureus strains were lower to those observed in
Higher levels of total IgG were observed in the mammary quarters plates sensitized with strain 806 (NP).
inoculated with strain 806 (NP) than in the control quarters at all In plates sensitized with strain 806 (NP), a significant effect of
sampling times evaluated (p < 0.05). In mammary quarters inoculated S. aureus challenge was observed over time finding differences (p =
with strain 5011 (P) the total IgG levels did not differ from control 0.039) in the IgG2 levels between the different experimental groups
quarters and quarters inoculated with strain 806 (NP). (Fig. 5C). In mammary quarters inoculated with strain 806 (NP) a sig
In plates sensitized with strain 5011 (P), a significant effect of nificant increase was observed at d 7 and 21 pi compared with control
S. aureus challenge was observed over time finding differences (p = quarters (p < 0.05). In mammary quarters inoculated with strain 5011
0.015) in the total IgG levels between the different experimental groups (P) a significant increase was observed on d 21 pi compared with control
(Fig. 5D). In mammary quarters inoculated with strain 806 (NP) higher quarters (p < 0.05).
total IgG levels were observed at all sampling times evaluated (p < 0.05) In plates sensitized with strain 5011 (P), a significant effect of
compared with control quarters. In mammary quarters inoculated with S. aureus challenge was observed over time finding differences (p =
strain 5011 (P) total IgG levels did not differ from control quarters and 0.005) in the IgG2 levels between the different experimental groups
quarters inoculated with strain 806 (NP). (Fig. 5F). In both mammary quarters inoculated with strain 806 (NP)
In plates sensitized with strain 806 (NP), a significant effect of and strain 5011 (P) a significant increase was observed at d 7 pi and
S. aureus challenge was observed over time finding differences (p = continue until d 21 pi compared with control quarters (p < 0.05). The
0.011) in the IgG1 levels between the different experimental groups IgG2 levels for both S. aureus strains in plates sensitized with strain 5011
(Fig. 5B). In mammary quarters inoculated with strain 806 (NP) higher (P) were higher than the levels found in plates sensitized with strain 806
levels of IgG1 were observed at all sampling times evaluated (p < 0.05) (NP).
compared with control quarters. In mammary quarters inoculated with
strain 5011 (P) a significant increase was observed at d 14 pi compared 4. Discussion
with the levels of control quarters and was maintained until d 21 pi (p <
0.05). This study focused on two S. aureus isolates collected from bovine
In plates sensitized with strain 5011 (P), a significant effect of IMI with different adaptation genotypes (low and high) to the bovine
S. aureus challenge was observed over time finding differences (p = MG using an experimental challenge model for bovine mastitis to gain
0.018) in the IgG1 levels between the different experimental groups insights into the mechanism of the immune response induced by distinct
(Fig. 5E). In mammary quarters inoculated with strain 806 (NP) a sig strains of this organism. In vitro studies of host-pathogen interactions
nificant increase was observed at d 7 and 21 pi compared with control have demonstrated that differences exist between S. aureus strains and
quarters (p < 0.05). In mammary quarters inoculated with strain 5011 lineages in their ability to invade and/or survive intracellularly and to
(P) a significant increase was observed at d 7 and 21 pi compared with elicit expression of pro-inflammatory mediators in bovine mammary
8
cells [16,28,29]. In a recent study bovine experimental IMI using was isolated [27]. In all quarters challenged with this strain, only small
S. aureus strains belonging to the main bovine lineages detected in lumps in milk were observed on day 14 pi without presence of local
Ireland (CC97 and CC151) were performed inducing a differential im inflammatory signs and coinciding with the highest SCC values. Our
mune response in the host demonstrating that the outcome of mastitis results agree with previous studies where high SCC were reported even
induced by this pathogen was strain dependent [19]. CC 97 has been when S. aureus was not isolated from the milk of the challenged MG [39,
identified as the most prevalent genotype around the world [30]. 46]. The fact that S. aureus could not be isolated at all sampling periods
However, a wide variety of genotypes have been detected in different after challenge with both strains reflects the previously described
countries, as well as in regions within each country [30]. Although some cyclical shedding pattern of S. aureus in milk [45,47]. Collectively these
genotypes are considered to have higher pathogenic potential, a clear findings demonstrated that, although both S. aureus strains were capable
link between presence of virulence factors and clinical outcome or of establishing an IMI, strain 806 (NP) triggered a more rapid and
mastitis severity has not been established [30,31]. In this study, we used intense immune response than strain 5011 (P) at early stages (24–72 h
two isolates from bovine mastitis cases with different genotypic profiles pi) persisting until day 7 pi; while strain 5011 (P) multiplied initially at a
characterized by microarray analysis as unusual bovine lineages: strain lower rate but from 7 d pi to the end of the study was detected more
806 (NP) that belonged to ST350-MSSA and is a rare ST that has been frequently in milk probably associated to an increased ability to evade
isolated from humans, dogs, horses, a wild deer and cows with mastitis host defenses and adapt to the MG environment.
(Monecke, S; data not published) and strain 5011 (P) that belonged to The differences observed in this study on the persistence of S. aureus
CC188-MSSA and is considered mostly a human lineage although it has strains in the MG could be related to the carriage and expression of
also been found in association with bovines [32,33]. The ST used in the certain virulence factors, as shown by Buzzola et al. [48] in experimental
present study have also been detected in bovine milk in other countries IMI in murine MG challenged with S. aureus strains of different agr type.
[30]. Although direct comparisons cannot be made with the study by These authors, demonstrated that S. aureus agr-type II or IV strains were
Niedziela et al. [19] since different lineages of S. aureus were used, both more efficiently eliminated from the MG than those of type I, suggesting
studies agreed that the genetic characteristics of the strains were asso that agr-type I strains may persist in greater numbers in mammary tissue
ciated with differential local immune response that determined the than agr types II, III and IV strains. In the present study, strain 806 (NP)
course and severity of the infection. classified as agr-type II was eliminated from MG with greater efficiency
In the last decades, experimental in vivo infection models have been than strain 5011 (P) (agr-type I), which evaded the immune system and
extensively used and constitute an effective tool for the investigation of persisted in the MG until 56 days pi. These results are in agreement with
the host immune response against S. aureus causing bovine mastitis [22, previous research from our laboratory [20], in which the immune
34–38]. Although animal conditions and experimental designs vary response induced in mice MG challenged with two S. aureus strains
between studies, experimental challenges with S. aureus generally range isolated from bovine mastitis with different phenotype, genotype and
from mild or moderate acute clinical to subclinical mastitis depending adaptation to the MG (P and NP) was evaluated. The S. aureus strain
on the number of challenge organisms and the anatomical inoculation isolated from a NP IMI showed a greater ability to multiply in mammary
site [39–41], triggering a slight local immune reaction of the MG and tissue in the early stages of the IMI compared with the P strain, while the
generally no systemic involvement. As a result, these experimental in P strain multiplied initially at a lower rate, but increased its replication
fections may very often become persistent [35,42]. Besides, the S. aureus capacity from 120 h pi to the end of the study (11 days pi), indicating a
strain effect on clinical/sub-clinical mastitis has recently been demon greater ability to evade the immune system and thus persist in the MG
strated [19,43,44]. [20]. In addition to the agr type, the cap gene type and capsular poly
There are numerous experimental IMI studies in cattle in which saccharide (CP) expression has also been shown to play an important
different S. aureus strains and bacterial concentrations have been used: role in S. aureus intracellular survival. In this context, in in vitro studies
~40 CFU/ml [39]; ~300 CFU/ml [24]; ~1000 CFU/5 ml [18]; ~2000 with bovine mammary epithelial cells, Bardiau et al. [13] demonstrated
CFU/ml [34]; 5 × 104 CFU/2 ml [36]. In the present study, a bacterial that isolates belonging to agr group II, cap8 positive and expressing CP8,
suspension containing ~400 CFU/ml was used. The selection of the were less likely to survive intracellularly than isolates belonging to agr
bacterial concentration was based on previous studies in which using a group I not expressing any CP. In our study, although both S. aureus
low number of bacteria (~72 CFU of the S. aureus strain Newbould 305 strains carried cap genes, the expression of CP5 or CP8 in vitro was not
in 2 ml) in mid-lactation cows with low SCC a mild clinical IMI in all determined, therefore direct comparisons with previous research [13]
inoculated quarters was established [39]. We also selected this con cannot be made.
centration to avoid overwhelming the MG immune system. In this study, The SCC has long been used as an indicator of inflammation for
using MGs with low SCC, S. aureus experimental IMI were successfully bovine mastitis diagnosis based on the increase in the number of cells
established for both strain. Detection of S. aureus in milk from chal due to the infiltration of neutrophils that gain access to the milk as a
lenged quarters had the characteristics of intermittent and cyclical consequence of the inflammation [49]. In experimental challenges with
shedding [45] and bacteria were not detected simultaneously in all S. aureus the time to the appearance of detectable signs of mastitis is
challenged quarters at a given sampling time. In the quarters challenged variable depending on the inoculum size and cow factors and can be
with strain 806 (NP), bacteria were recovered from milk at 24–72 h pi in limited to a gradual increase in the SCC over a period of 48–72 h after
half of the challenged quarters. However, in this period the highest SCC challenge, with often concomitant isolation of S. aureus in milk [39–41,
were detected in all challenged quarters irrespective of the bacterio 50]. In this study, the SCC response elicited by the two S. aureus strains
logical status, reaching a mean of 3.7 × 106 cells/ml at d 2 pi. The lack of differed and showed variations over time. In mammary quarters inoc
isolation in four of the eight challenged quarters could have been due ulated with strain 806 (NP) the inflammatory response induced was
both to the contribution of the innate immune response to eliminate the greater and earlier than the one induced by strain 5011 (P), since a SCC
inoculum and to the presence of organisms in milk below the detection peak was observed at d 2 pi, while in mammary quarters inoculated with
limit of the methodology used. From d 28 to d 56 pi this S. aureus strain strain 5011 (P) no variations in SCC were observed until d 4 pi reaching
was no longer detected in milk of challenged cows. All mammary the maximum values at d 14 pi; indicating a lower and delayed initial
quarters challenged with strain 806 (NP) developed mild clinical inflammatory response. This delay in response to strain 5011 (P)
mastitis (score 1) between 1 and 7 d pi and high SCC during this period. compared with strain 806 (NP), could be associated both with (1) less
In quarters challenged with strain 5011 (P), bacteria were recovered initial stimulation of macrophages to release cytokines and recruit
from milk from d 7 pi and up to d 56 pi in one of the challenged quarters, neutrophils to the MG minimizing the inflammatory response and (2)
confirming the ability of this strain to adapt to the MG microenviron increased capacity to survive intracellularly. Differential adhesio
ment and develop a persistent IMI as in the natural case from which it n/internalization and intracellular persistence capacities were observed
9
for both S. aureus strains in mammary epithelial cells in a previous in and size, site of inoculation, lactation period and other cow factors as
vitro study [16]; in which strain 5011 (P) showed higher adhesio well as sensitivity of the ELISA. In this study, the IL-1β response
n/internalization and persistence capacity in MAC-T cells and greater depended on the S. aureus strain infused. In mammary quarters inocu
resistance to microbicidal mechanisms than strain 806 (NP) [16]. lated with strain 806 (NP) IL-1β concentration in milk increased sharply
Internalization might protect bacteria from clearance by the immune at d 2 pi reaching a peak and remained at higher levels than those
system and allow for long-term persistence in chronically infected hosts detected for strain 5011 (P) or control during the observation period.
[51]. Accordingly, it is possible that induction of inflammatory During the inflammatory response, IL-1β regulates the expression of
response, as indicated by a noticeable increase in milk SCC from d 4 pi adhesion molecules in epithelial cells and the chemotaxis of neutrophils
occurred once the strain 5011 (P) reached a minimum threshold to in the early stages of infections caused by S. aureus [60]. The high levels
trigger recognition by the innate immune system. Although this period of IL-1β induced by strain 806 (NP) were associated with the highest SCC
of delay to trigger the immune response could be associated with the detected, demonstrating the ability of this strain to induce a rapid local
greater capacity of this strain to internalize in mammary cells in vitro, inflammatory response, which in turn contributed to control the
the experimental setting of the present study does not allow to confirm multiplication of the microorganism. In mammary quarters inoculated
this hypothesis. Results obtained in this study are indicative that the SCC with strain 5011 (P), although higher IL-1β levels were detected at day 4
response was directly influenced by the characteristics of the S. aureus pi, concentrations did not differ from those found in control quarters at
strain and are in line with previous findings from Niedziela et al. [19]. every sampling time. This strain induced a lower and more gradual local
Previous studies have indicated that NO is a key mediator of the inflammatory reaction, demonstrated by the low levels of IL-1β and NO
inflammatory responses caused by IMI [52]. Atakisi et al. [53] have in milk, as well as by the low SCC detected until day 4 pi.
observed higher concentrations of NO in milk from MG with subclinical IL-6 is considered one of the key mediators of the acute phase
mastitis compared with non-infected MG, indicating a relationship be response in inflammation [61]. In addition, this cytokine is involved in
tween elevated levels of NO and inflammation. In agreement with Ata differentiation, activation of lymphocytes and production of immuno
kisi et al. [53], the highest levels of NO detected in milk from quarters globulins [62]. Increased concentrations of IL-6 have been detected in
inoculated with both S. aureus strains were associated with the time milk and blood of cows with naturally acquired [63] or experimentally
periods when highest SCC were detected. The ability of macrophages to induced mastitis during lactation [64] and in mammary secretion of
kill microbial pathogens has been linked to their capability to generate chronically infected S. aureus quarters during involution [12]. In this
NO, a highly bactericidal moiety [54]. In the present study, the highest study, in mammary quarters inoculated with strain 806 (NP) two IL-6
concentrations of NO observed at d 3 pi in quarters challenged with peaks on days 2 and 14 pi were observed. IL-6 plays a crucial
strain 806 (NP) could have contributed to the faster bacterial clearance anti-inflammatory role in both local and systemic acute inflammatory
of the MG compared with strain 5011 (P). Levels of NO and SCC began to responses by controlling the level of pro-inflammatory, but not
increase from day 4 prior to the detection of the organisms in milk and anti-inflammatory cytokines [65]. The first increase on day 2 may have
remained high until day 21 in quarters challenged with strain 5011 (P). been due to an inflammatory effect since it coincided with the high SCC,
This suggests that strain 5011 (P) weakly stimulated macrophages which would indicate a role in the recruitment of cells to the site of
during the first 4 days inducing a more gradual and delayed inflam infection. In murine experimental models, when the inflammation re
matory reaction than strain 806 (NP), favoring the adaptation of the solves, the recruited leukocyte population shifts primarily from neu
microorganism to the microenvironment of the MG and further estab trophils to monocytes and this transition is regulated by IL-6 [66]. In the
lishment of IMI. present study, the second increase on day 14 could be related to an
Levels of Lf in milk vary according to the age of the cow, stage of anti-inflammatory effect which coincided with an abrupt decrease in
lactation, parity, SCC and presence of pathogenic organisms [55]. SCC. Additional studies are necessary to confirm the association of these
Hagiwara et al. [55] have reported Lf levels in milk samples from cytokines and their action as part of the innate immune response against
healthy quarters and with subclinical mastitis of approximately 170 and infection. In mammary quarters inoculated with strain 5011 (P), IL-6
501 μg/ml, respectively. In the present study, in accordance with the concentrations were similar to those found in the control quarters.
aforementioned, maximum Lf concentrations were detected in mam Hagiwara et al. [63] confirmed high levels of IL-6 in the first stage of
mary quarters inoculated with strain 806 (NP) on d 3 pi (average natural infections, with a mean concentration of IL-6 on the first day of
maximum value ~375 μg/ml). In mammary quarters inoculated with the disease 25 times higher in milk samples and 5 times higher in serum
strain 5011 (P) the maximum Lf concentrations were detected on d 21 pi samples in cows with acute clinical mastitis compared with normal
(average maximum value ~285 μg/ml), while in control quarters Lf cows. These authors observed that sera and whey samples from cows
concentration did not vary through the experimental period (~120 infected with E. coli, K. pneumoniae, S. aureus or Streptococcus sp. con
μg/ml). During inflammation, Lf production by MG epithelial cells is not tained significantly higher concentrations of IL-6 than those of normal
only intensified, but also released from neutrophils secondary granules cows, suggesting that the levels of IL-6 in milk and serum depend not
[56]. Lactoferrin increases production of inflammatory cytokines and only on the stage of infection but also on the type of microorganisms that
chemokines, and migration of leukocytes to the MG [57]. Kawai et al. causes mastitis. In agreement with these findings, the variation in levels
[58] demonstrated that high Lf concentrations in mastitis milk were of IL-6 detected in the present study could have been due to the inherent
associated with the severity of the mammary inflammation. In this study characteristics of the two S. aureus strains. However, further studies are
the highest levels of Lf detected in milk from quarters inoculated with needed to elucidate the mechanisms of IL-6 production and action in the
both S. aureus strains coincided with high SCC in the same time periods, MG infected with different strains of the same pathogen.
indicating an association with the magnitude of inflammation. IL-4 is a multifunctional pleiotropic cytokine produced mainly by
The IL-1β response during experimental IMI has been shown to be activated T cells, but also by mast cells, basophils and eosinophils.
highly variable compared to other cytokines. Following an experimental Functionally, IL-4 is best known for defining the Th2-type response
infection of mid lactation cows with ~40 CFU of S. aureus strain New profile of CD4 T cells and for regulating cell proliferation, apoptosis, and
bould 305, Banneman et al. [39] observed an increase in the IL-1β expression of numerous genes in various cell types [67]. In this study,
concentration in milk after 32 h which was maintained for an additional mammary quarters inoculated with strain 806 (NP) showed a significant
8 h. Riollet et al. [59], after experimental inoculation of cows in mid increase on d 1 pi, a second increased on d 7 pi and higher levels than the
lactation with ~100 CFU of a S. aureus strain isolated from a natural other experimental groups until d 21 pi. Previous research from our
bovine mastitis case (strain 107-59), did not detect this cytokine in milk laboratory [12] showed significantly higher IL-4 levels in mammary
at any of the evaluated times (from d 1 to d 28 pi). Differences in results secretions from quarters chronically infected with S. aureus compared
could have been related to strain characteristics, inoculum preparation with control quarters at 24 h post-drying off, with a decrease until day
10
14 and a return to high levels at day 21 of involution. IL-4 participates in cells. However, highest levels of specific opsonic IgG2 are expected to be
the alternative activation of macrophages towards a type 2 tolerogenic achieved during inflammation 6–12 h before the peak neutrophil
profile (M2) to counteract inflammation through the release of IL-10 and response [72], which could be effective in previously immunized rather
transforming growth factor (TGF)-β, promoting wound healing and tis than in naïve animals. In addition, clearance of strain 806 (NP) took
sue repair [68]. To the best of our knowledge, there is no direct evidence place in most inoculated quarters mainly before day 14 pi; while
available about the role of IL-4 in S. aureus bovine mastitis [69] and no clearance of strain 5011 (P) did not occur in most of the inoculated
studies have quantified the IL-4 concentration in milk of an experi quarters. Therefore, the value of IgG2 for the clearance of bacteria in this
mentally induced S. aureus mastitis up to 21 d after challenge. In the study is doubtful. Although an adaptive humoral immune response was
present study, the high levels of IL-4 detected in milk from quarters developed against challenge with both S. aureus strains, a more effective
inoculated with strain 806 (NP) on d 1 pi, previous to peak concentra total IgG and IgG1 response was induced by strain 806 (NP), while strain
tions of both IL-1β and IL-6 is difficult to explain within the general 5011 (P) was more effective in generating an IgG2 response (even
knowledge of the functions of this IL [67,69]. Further studies are needed against the heterologous strain).
to evaluate other anti-inflammatory cytokines (IL-10, TGF-β) that could In this study, the sensitization of the ELISA plates to evaluate the
contribute to elucidate the anti-inflammatory role of IL-4 in bovine levels of immunoglobulins was carried out with lysates of the S. aureus
experimental infections by S. aureus. The second increase in IL-4 levels strains 806 (NP) and 5011 (P), observing similar results in the levels of
observed at d 7 pi coincided with high SCC in milk and could be specific IgG for both antigens in the different sampling times evaluated.
explained as an attempt to counteract the inflammation caused by This demonstrates cross-immunity against the antigens used, indicating
S. aureus in this period. This coincides with an increase in IL-6 levels to that immunoglobulins generated against one strain of S. aureus could
reach a peak at d 14 pi acting together with IL-4 modulating the in respond against other heterologous strains. To confirm these findings,
flammatory process, exerting an anti-inflammatory role. However, this functionality tests of the generated antibodies should be performed.
putative association needs to be confirmed by further studies. On the
other hand, in quarters inoculated with strain 5011 (P), an increase in 5. Conclusion
IL-4 levels was observed on day 1 pi, while at the rest of the evaluated
times the levels of this cytokine were similar to those found in the The present in vivo study confirms previous in vitro observations
control quarters. Bochniarz et al. [70], in a study in Holstein-Friesian about differential behavior of S. aureus strains with distinct adaptation
cows during lactation, determined that the concentration of IL-4 was capabilities to the MG demonstrating their ability to trigger, modulate
significantly lower in both serum and milk from cows with mastitis and evade the host immune response influencing the course and severity
caused by coagulase-negative staphylococci compared with control an of IMI. These features should be taken into account both in the diagnosis
imals, highlighting the importance of type and characteristics of path of intramammary infections within the framework of the classic control
ogen on the inflammatory response induced in MG. For all the programs for this organism, and in the design of experimental studies
aforementioned, additional studies with a greater number of animals are aimed at generating new control alternatives.
needed to characterize and understand the functions of IL-4 in the im
mune response to S. aureus. Ethics approval
IgG is the main effector of the humoral immune response of the MG
responsible for promoting phagocytosis of neutrophils [50]. The IgG1 All procedures with animals were conducted under protocols
subclass is the predominant type of antibody in milk from healthy approved by the Ethics and Security Committee of the Facultad de
quarters due to its selective transfer across the blood-mammary barrier Ciencias Veterinarias, UNL (protocol No 293) and were consistent with
[71]. In mastitic milk IgG2 becomes the dominant antibody subclass and the Guide for the Care and Use of Agricultural Animals in Agricultural
is considered the major opsonin that supports neutrophil phagocytosis in Research and Teaching (Federation of Animal Science Societies, 2010).
bovine MG [72]. In the present study, in both plates sensitized with
strains 806 (NP) and 5011 (P), the highest values of total IgG were CRediT authorship contribution statement
observed in mammary quarters inoculated with strain 806 (NP). In
mammary quarters inoculated with strain 5011 (P) values of IgG did not Carolina Engler: Writing – original draft, Methodology, Conceptu
differ from the other experimental groups. The possible explanations for alization, Bibiana Dallard, Writing – review & editing, Supervision,
these finding are that (1) immunoglobulins could be opsonizing the Resources, Funding acquisition, Conceptualization. María S. Renna:
bacteria present in milk leading to a decrease in detection of free im Writing – original draft, Methodology, Conceptualization. Camila
munoglobulins and (2) considering that in previous studies the strain Beccaria: Visualization, Methodology, Investigation. Paula Silvestrini:
5011 (P) showed a high capacity for adherence/internalization and Visualization, Methodology, Investigation. Silvana I. Pirola: Visuali
persistence in MAC-T cells [16] and that intracellular S. aureus can curb zation, Methodology. Elizabet A.L. Pereyra: Visualization, Methodol
the immune response of the MG [73], the reduction in specific IgG levels ogy. Celina Baravalle: Validation, Software, Methodology. Cecilia M.
in quarters challenged with 5011 (P) strain reflects a lower capacity of Camussone: Validation, Software, Methodology. Stefan Monecke:
this strain to stimulate a humoral immune response. Software, Methodology, Data curation. Luis F. Calvinho: Writing – re
In the case of IgG1 in both plates, sensitized with strains 806 (NP) and view & editing, Supervision, Conceptualization.
5011 (P), a significant increase was observed in mammary quarters
inoculated with strains 806 (NP) and 5011 (P) compared with control Declaration of competing interest
quarters. However, the values of IgG1 for both strains (NP and P) in
plates sensitized with strain 5011 (P) were lower than the values from The authors declare that they have no known competing financial
plates sensitized with strain 806 (NP). In the case of IgG2 in both plates, interests or personal relationships that could have appeared to influence
sensitized with strains 806 (NP) and 5011 (P), a significant increase was the work reported in this paper.
observed in mammary quarters inoculated with strains 806 (NP) and
5011 (P) compared with control quarters. However, the values of IgG2 Acknowledgments
for both strains (NP and P) in plates sensitized with strain 806 (NP) were
lower than the values from plates sensitized with strain 5011 (P). These The authors express their appreciation to Mr. Gaston Reibel and Mr.
results could indicate a specific humoral immune response against José Maria Copes for field technical assistance. This work was supported
S. aureus during the experimental challenge that could have contributed by Argentine National Agency for the Promotion of Science and Tech
to opsonophagocytosis and the elimination of bacteria by phagocytic nology (ANPCyT) (PICT 2016–1798 and PICT 2019–3428).
11
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13

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Microbial Pathogenesis 172 (2022) 105789

Contents lists available at ScienceDirect

Differential immune response to two Staphylococcus aureus strains with

Figure 1. Schematic overview of the S. aureus intra

3. Results 3.2. Bacteriological examination, SCC and cell microscopic observation

806 (NP) (Animal 1 (− ) (− ) 5000 1500 500 (− ) (− ) 100 300 (− ) (− ) (− ) (− ) (− )

Fig. 3. A) Nitrite concentration (generated by spontaneous oxidation of NO). B)

3.4. Cytokine levels

IL-1β, IL-6 and IL-4 concentrations were evaluated in milk samples

Fig. 5. A) Total IgG, B) IgG1 and C) IgG2. Assays

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Engler 2022

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Engler 2022

Uploaded by

Document Information

Original Title

Copyright

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Share this document

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Did you find this document useful?

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Copyright:

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Engler 2022

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Copyright:

Available Formats

Microbial Pathogenesis 172 (2022) 105789

Contents lists available at ScienceDirect

Differential immune response to two Staphylococcus aureus strains with

Figure 1. Schematic overview of the S. aureus intra­

3. Results 3.2. Bacteriological examination, SCC and cell microscopic observation

806 (NP) (Animal 1 (− ) (− ) 5000 1500 500 (− ) (− ) 100 300 (− ) (− ) (− ) (− ) (− )

Fig. 3. A) Nitrite concentration (generated by spontaneous oxidation of NO). B)

3.4. Cytokine levels

IL-1β, IL-6 and IL-4 concentrations were evaluated in milk samples

Fig. 5. A) Total IgG, B) IgG1 and C) IgG2. Assays

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Figure 1. Schematic overview of the S. aureus intra