periparturient immune function and the acute phase … · the early defense against intramammary...
TRANSCRIPT
“What we have lost? Mastitis resistance in local breeds ”
Periparturient Immune Function and the Acute Phase Response in Dairy Animals , Rome, September 8-9, 2016 Massimo AMADORI Laboratory of Cellular Immunology, IZSLER, Brescia
April 14th, 2016
F. Riva1, B. Castiglioni2, M.F. Addis3, E. Trevisi4, M. Amadori5, C. Pollera1, J.F. Felipe-Soares1, G. Curone1, D. Vigo1, P. Moroni1, V. Bronzo1 , P. Cremonesi2
1 Department of Veterinary Medicine, University of Milan, Italy; 2 Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Lodi, Italy; 3 R&D - Proteomics Lab, Porto Conte Ricerche S.r.l., Alghero, Italy; 4 Istituto di Zootecnica, Facoltà di Scienze Agrarie, Alimentari ed Ambientali, Università Cattolica del Sacro Cuore, Piacenza, Italy; 5 Laboratory of Cellular Immunology, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy.
Holstein Friesian cattle: too many culled cows !
IMI !!
Holstein dairy cows: reduced environmental fitness
High-yielding dairy cattle, potential ≥ 10,000 kg/lactation
Clear correlation with mastitis , and also ketosis and
lameness (Ingvartsen et al., 2003). Immunosuppression – Dysregulation of the
inflammatory response.
Innate immune responses to metabolic stress
Challenges to homeostasis (acidosis, osmolarity changes, hypoxia, ROS, ATP/AMP, a.a.) NEFA TLR4
inflammasome IL-1b / IL18
P38 MAPK
TLR ligands, cytokines, physico-chemical stressors
Pi3 / Akt / mTOR
Expression of IL-12 and IL-10 in myeloid cells
Regulation of pro and anti-inflammatory
responses in tissues
eIF2a
Persisting welfare problems, serious repercussions
Poor housing conditions in the dry period
• Reduced space allowances (< 8m2) in loose cattle housing
• Poor cleanliness
• Poor removal and replacement of litter
• Low-quality feeds (over and underconditioned cows at calving).
POOR COPING WITH ENVIRONMENTAL STRESSORS !
Prevalence of mastitis
• 1,287 herds of 5 Italian regions, from 2012 to 2016 • 90-95% of animals: Holstein Friesian cows • 4.81% farms: > 80% of cows treated for clinical mastitis • 27.20% farms: 40-80%
(Bertocchi and Fusi, Italian National Animal Welfare Reference Centre)
NY State (USA): 40% on average
(Moroni P., personal communication)
Local cattle breeds: better animal health data
Lower disease prevalence, reduced culling rates, high mean parity levels. Italian Rendena breed (alpine area)
Dual purpose cattle breed, yields around 5,000 kg / lactation, life expectancy > 10 years, alleged prevalence
of infectious mastitis < 4 %. Is it a matter of innate immunity ?
Bovine mammary epithelial cells: a foundation of protection
• bMEC lining the inner surface of the mammary gland are crucial for the early defense against intramammary pathogens.
• They constitute a physical barrier. • They produce several antimicrobial substances and inflammatory
mediators. • TNF-alpha, interleukin (IL)-1beta, GM-CSF, IL-8, RANTES , lactoferrin
(Lf) and serum amyloid A (SAA), cyclooxygenase-2 (COX-2) (Zbinden C. et al., 2014, PLoS ONE 9(1): e87374)
Innate
immune
defences
(epithelial
cells)
Inflammatory response
Infection is not controlled
Failure of epithelial cells: granulocytes go into action !
Fundamental issues
• What underlies mastitis resistance in local breeds ?
• How can we investigate it ?
• Can we define markers of innate immune response in the mammary gland discriminating low from high-yielding dairy cattle ?
Our experimental farm
• High and low-yielding dairy cattle reared together
• 6 Holstein Friesian. 3 Rendena Breed. 3 Brown Swiss. 1 Italian Pezzata Rossa. 1 Italian Grey Alpine.
• Mean parity: 4 ± 1
• No antibiotic treatments at dry-off
• Four milk and blood samplings: T1=dry-off, T2=1 day after calving, T3=7-10 days after calving, T4=30 days after calving, T5=60-70 days after calving.
• Milk sampling after cleaning and teat disinfection
• Milk of each quarter separately collected (4 samples / cow)
Our mixed, happy herd !
Data sets
• Blood: clinical immunology and chemistry on plasma samples (pos and neg APP, energy metabolism)
• Milk: lysozyme, expression of cytokine genes and marker genes of leukocytes and epithelial cells, bacteriological analyses and determination of SCC, metagenomic analysis for 16S rRNA gene on bacterial DNA.
• A) between time points B) between breeds.
• Caveat: no final conclusions, we propose a model and a methodology.
Time points differences, only.
BLOOD
Time point
differences,
only
Time point
differences, only
A significantly higher concentration in Friesan cows at T3
Time point
difference in
Friesan cows,
only (T3)
PECULIARITIES OF FRIESIAN
COWS
Significant breed difference at dry-off (T1): a
greater mass of skeletal muscle in autoctonous
cattle at the end of lactation ?
Ceruloplasmin: significant and consistent
breed differences at T1, T4, T5
(peak and later phase of lactation)
Higher constitutive expression ?
Most important!
Time point and breed differences at T2
Time-course reminiscent of bilirubin levels!
Bilirubin ?
Milk lysozyme: positive modulation of innate immunity
in the colostral phase, much higher
in local breeds
Distribution of SCC (cellof milk)
Do fewer cells in colostrum of Friesian cows account for the difference ? NO !
Gene expression studies on milk cells
• Total milk cells of 50-ml samples, lysed in 3 ml of TRI Reagent
• Milk fat globules: 0.5-ml milk fat samples were lysed in 1.5 ml TRI reagent
• Expression of inflammatory cytokine genes
• Infiltration of leukocytes (expression of CD45 gene)
• Presence of epithelial cells (KRT5 gene)
• GAPDH as housekeeping gene
• Delta Ct values multiplied by 10,000 (Arbitrary Units of expression in the sampling volume)
Friesian vs local breeds: gene expression in total milk cells
Bacteriological findings:
healthy udders, isolation of non-pathogenic bacteria
% culture-positive quarters
Friesian vs local breds: gene expression studies in milk fat globules:
Significant up-regulation in local breeds, only
Friesian vs. Rendena breed: gene expression studies on total milk cells
Total milk cells, Friesian vs. Rendena breed
The prevalence of leukocytes in colostrum is much higher in Friesian cattle !
250 150
100
75
50
37
25
20
15
10
Ho
lstein 2
42
2 (A
D) 1
:10
Ren
den
a 13
29
(PD
) 1:1
0
Bru
na 7
27
7 (P
S) 1:1
0
Bru
na 7
27
7 (A
D) 1
:10
Ren
den
a 13
29
(PS) 1
:10
Ren
den
a 13
30
(PS) 1
:10
Ren
den
a 13
30
(PD
) 1:1
0
Ren
den
a 13
30
(AD
) 1:1
0
Xanthine dehydrogenase/oxidase
Lactoferrin
Ig heavy chain
Caseins Ig light chain
Alpha lactalbumin
Beta lactoglobulin
Autochthonous breeds, especially Rendena, display a higher relative abundance of proteins with immune defense functions in colostrum
Same amount of milk loaded in each lane
T1 (post-partum)
Immune defense functions in colostrum (Brown, Friesian, Rendena)
0
10000
20000
30000
40000
50000
60000
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
Sign
al in
ten
siit
y
Relative front
Bruna 7227 (AD) 1:10
Bruna 7227 (PS) 1:10
Frisona 2422 (AD) 1:10
Rendena 1329 (PD) 1:10
Rendena 1329 (PS) 1:10
Rendena 1330 (AD) 1:10
Rendena 1330 (PD) 1:10
Rendena 1330 (PS) 1:10
25
0
15
0
10
0
75
50
37
25
20
15
10
Holstein 2422 (AD) 1:10
Rendena 1329 (PD) 1:10
Bruna 7277 (PS) 1:10
Bruna 7277 (AD) 1:10
Rendena 1329 (PS) 1:10
Rendena 1330 (PS) 1:10
Rendena 1330 (PD) 1:10
Rendena 1330 (AD) 1:10 Same amount of milk loaded in each lane
XD/XO
Lactoferrin
Ig heavy chain Ig light chain
Densitometry of bands
Fris
on
a 7
08
Fris
on
a 2
42
2
Ren
den
a 1
32
9
Ren
den
a 1
33
0
Bru
na
72
77
Bru
na
93
39
250 150 100
75
50
37
25
20
15
10
kDa
T3 (7-10 days)
tat
Fris
on
a 7
08
Fris
on
a 2
42
2
Ren
den
a 1
32
9
Bru
na
72
77
Bru
na
93
39
T4 (30 days)
250 150 100
75
50
37
25
20
15
10
kDa
As lactation proceeds, the relative abundance of major milk proteins is highly similar. Only a slightly higher protein concentration can be seen in all T3 samples. Higher resolution techniques, such as IEF or chromatography, will be required to highlight possible differences.
Same amount of milk loaded in each lane
Later phases: differences on the wane
T3 - Denst
Holstein 708
Holstein 2422
Rendena 1329
Rendena 1330
Bruna 7277
Bruna 9339
25
0
15
0
10
0
75
50
37
25
20
15
10
0
10000
20000
30000
40000
50000
60000
0 0,2 0,4 0,6 0,8 1
Sign
al in
ten
sity
Relative front
Bruna 7277
Bruna 9339
Frisona 708
Frisona 2422
Rendena 1329
Rendena 1330
Densitometry of bands: T3
0
10000
20000
30000
40000
50000
60000
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
Sign
al in
ten
sity
Relative front
Bruna 7277
Bruna 9339
Frisona 708
Frisona 2422
Rendena 1329
Holstein 708
Holstein 2422
Rendena 1329
Bruna 7277
Bruna 9339
25
0
15
0
10
0
75
50
37
25
20
15
10
t Densitometry of bands: T4
Microbiota Innate Immune System (a-defensins)
Co-evolution of PRRs and microbial colonization
Samples
Single quarters of 3 Rendena and 3 Friesian cows at 4 different
times (T1 to T4), with summation of the four sets of results.
Metagenomic Analysis of Milk: is milk microbiota different ?
DNA extraction
Microbial DNA was extracted as previously described (Cremonesi
et al., JDS, 2006).
Preparation of gene libraries
MiSeq
Sequencing
Analysis of results
Reference database
The 16S rRNA gene (region V3-V4) was
amplified according to the standard
protocol of Illumina gene libraries, and
sequenced on the Illumina MiSeq
platform (Illumina, San Diego, USA). Data
were analyzed by Miseq Reporter
software. The GreenGenes database was
used as reference for classification of
sequences.
Miseq Reporter
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
100,0
RENDENE
FRISONE
94%
2% 2% 1% 1%
Firmicutes
Bacteroidetes
Proteobacteria
Actinobacteria
OTHER
RENDENA breed: Phylum
65% 15%
11%
6%
1% 2%
Firmicutes
Proteobacteria
Actinobacteria
Bacteroidetes
Verrucomicrobia
OTHER
FRIESIAN breed: Phylum
• In both breeds Firmicutes (94% -
Rendena; 65% - Friesian) are the most
common Phylum
• In Friesian cows microbiota also
consists of Proteobacteria (15%),
Actinobacteria (11%) e Bacteriodetes
(6%).
Metagenomic analysis: phylum
In Rendena cows Streptococcus is highly prevalent (71%), followed by Lactobacillus
(10%) and Pediococcus (6%), whereas in Friesian cows the prevailing genera are
Streptococcus (29%), Lactobacillus (6%), Corynebacterium e Staphylococcus (4%).
71% 10%
6%
1%
1%
1% 1%
9% Streptococcus
Lactobacillus
Pediococcus
Staphylococcus
Leuconostoc
Lactococcus
Enterococcus
OTHER
RENDENA: Genus
29%
6%
4%
4% 3%
2% 2% 2% 2% 2% 2%
2%
40%
Streptococcus
Lactobacillus
Corynebacterium
Staphylococcus
Bradyrhizobium
Clostridium
Aerococcus
Bacteroides
Blautia
Alkaliphilus
Pediococcus
Bifidobacterium
OTHER
FRIESIAN: Genus
Metagenomic analysis: genus
Metagenomic analysis: species A highly significant difference in
the Streptococcus genus . In the
Rendena breed, 48% of
streptococci consist of
Streptococcus thermophilus as
opposed to 2% in Friesian cows !
Streptococcus thermophilus è a
lactic bacterium used in the
production of fermented milks ,
yogurts and many cheese types. Its
high concentrantions in Rendena
milk is conducive to high clot
yield and cheese production.
48%
23%
3%
2%
2%
2%
2%
2% 2% 1%
1%
12%
Streptococcus thermophilus
Streptococcus vestibularis
Streptococcus fryi
Pediococcus stilesii
Lactobacillus brantae
Lactobacillus japonicus
Lactobacillus camelliae
Lactobacillus paracasei
Pediococcus argentinicus
Staphylococcus xylosus
Streptococcus bovis
OTHER
18%
5%
3%
3%
3%
2%
2% 2%
1%
1% 1%
1% 1% 1%
56%
Streptococcus vestibularis
Streptococcus bovis
Streptococcus fryi
Bradyrhizobium pachyrhizi
Aerococcus viridans
Phascolarctobacterium succinatutens
Streptococcus thermophilus
Lactobacillus camelliae
Bacteroides denticanum
Alkaliphilus peptidifermentans
Facklamia tabacinasalis
Clostridium alkalicellulosi
Bifidobacterium pseudolongum
Lactobacillus brantae
OTHER
RENDENA: Species
FRIESIAN: Species
Take-home messages (caveat !)
• Mastitis resistance in local cattle breeds can be traced back to fundamental differences in the colostral phase.
• With respect to Friesian cows, the differences in the colostral phase are related to stronger innate immune responses in the mammary gland and to the lower early level of lipomobilization (NEFA).
• The differences in colostrum are not accounted for by lower concentrations of SCC in Friesian cows.
• Leukocytes of Friesian cows are probably less efficient in the colostral phase.
• The milk microbiota of a local breed shows highly significant differences from that of Friesian cows.
Indirect confirmation ex iuvantibus: higher SAA and fewer mastitis cases in IL 2-treated Friesian cows after calving
Zecconi et al. CIMID 2009, 32, 439–451
Treatment Days in milk
10-12 17-19 24-26 31-33
IL-2 79.5% 74.0% 72.7% 77.5%
Control 61.2% 66.7% 79.2% 74.0%
P 0.03 0.21 0.21 0.35
1
Healthy quarter frequency
IL-2 administration: a useful immunotherapy agent ?
Zecconi et al. CIMID 2009
45 subjects. 3 herds (115-230 lactating cows). 23 treated and 22 control subjects. Random allocation of quarters to treatment and control
1 dose (800 pg) s.c., area of the supramammary lymph node, days 3-5 after calving.
Significantly higher frequency of healthy quarters over 2 weeks after calving in IL-2 treated cows.
0
10
20
30
40
50
60
70
80
90
100
Str.agal S.aur. Str.amb. SCN
Nulla
Amoxi
Cefa I
Cloxa
Peni
Pirli
Wilson, 1999
Y axis: % curing rates . Red bars: no antibiotic treatment. SCN: coagulase-negative staphylococci
The innate immune response works !!
Hypotheses and future work
• High NEFA levels and other factors after calving could inhibit in Friesian cows a badly needed local innate immune response in the colostral phase
• This response would prime the epithelia of the mammary gland to an effective and time-limited response to mastitis agents.
• Also, non-infectious stressors in the dry phase could negatively affect such a response («memory» function of the innate immune system).
• The later innate responses of epithelial cells in Friesian cows would be often less effective and often poorly controlled.
• As a result, leukocytes could colonize more easily the mammary gland in Friesian cows vis-à-vis common bacterial pathogens.
An informal research consortium based on different types of expertise and deep mutual trust
F. Riva, C. Pollera, J.F. Felipe-Soares, G. Curone, D. Vigo, P. Moroni, V. Bronzo: microbiology and molecular diagnostics
B. Castiglioni, P. Cremonesi : metagenomics
M.F. Addis: proteomics
E. Trevisi: zootechnics, clinical chemistry
M. Amadori: veterinary immunology
Thank you for
the attention !