“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 !