Update on Staphylococcus aureus Mastitis

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John R. Middleton

College of Veterinary Medicine,

University of Missouri, Columbia

Staphylococcus aureus

• Gram-positive, facultatively anaerobic, non-motile, non-sporulating, catalase positive, coagulase-positive, hemolytic coccus.

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Staphylococcus aureus

• Most prevalent contagious pathogen

• Most often subclinical– Milk quality

– Milk yield

• Chronic infections – Intermittent clinical flare-ups

• Fluctuating SCC and bacteria counts

• Occasional severe (Toxic) mastitis

• Variable response to treatment

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1961

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“The increase in the importance of staphylococcal mastitis since the advent of the antibiotics and the fact that many of these organisms are resistant to penicillin have caused some anxiety to those interested in milk production.”

1961

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“Quantitative analysis suggests that most infections of other sites were secondary to infection of the udder.“

The 5-Point Plan - 1969

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“Mastitis has been and continues to be the most costly dairy cattle disease…”

“A true mastitis control program must be directed towards prevention…”

S. aureus Mastitis

• Implementation of the 5-point plan has led to a sizeable reduction in S. aureus mastitis on many farms.

• Individual herds can still be plagued with the disease and, depending on chronicity and transmissibility within herd, it can be difficult to eradicate.

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Pathogenesis

S. aureus

Mammary epithelial cell

Fnbp or direct cell to

cell interaction

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Evasion of Host Defenses

S. aureus

Casein

Fat

Low Ab

PMNSpA

Capsule

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Pathogenesis

• Once established in gland– Ulceration and erosion of tissues– Tissue infiltration with inflammatory cells– Damage to secretory epithelial cells leading to occlusion of

ducts & alveoli, trapping S. aureus– Granulomas and microabscesses– Nidus of infection for other parts of gland

• Produces numerous toxins and enzymes – Role?

• Superantigens• Tissue damage

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Canal

Streak Canal and lining of

Keratin

Environmental

Contagious

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Sources of Infection

67%

Lact. Pop.

67%

71%

Cows @

Part.

Heifers @

Part.

47%Heifers

@

Part.

Roberson et al.

39%

Roberson et al.

70%

Heifer

Udder

Skin

Cows @

Part.

Cows @

Part.

13%

Heifers @

Part.18%

Heifer

Udder

Skin

Heifer

Udder

Skin

Flies

• Haematobia irritans

– Capable of transmitting S. aureus to heifers

– Owens et al., 1998

• Herds that practiced effective fly control

– Lower risk of early lactation contagious pathogen IMI in heifers

– Nickerson et al., 1995

– Piepers et al., 2011

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Epidemiology

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λ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 λ

Epidemiology

• Study of NW U.S. dairies with a prevalence of S. aureus IMI ≥ 10%

– 43 strains

– 38/43 (88.4%) Unique to one herd

– 5/43 (11.6%) found in >1 herd

– 7/8 (87.5%) herds had >1 strain

– 6/8 (75.0%) herds had a predominant strain type• Accounted for 53-100% of isolates

– Milk SCC and NAGase did not differ between strains (P ≥ 0.046) within herd.

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Influence of Strain on Milk YieldCow ID Mean kg Milk Mean kg Milk

Control Quarter Infected Quarter

Group 1:

75 1.29 0.45

97 6.58 3.49

153 3.86 2.29

164 1.56 0.00

201 1.95 0.77

209 2.11 0.00

226 5.40 4.04

353 2.47 0.00

3.15 * 1.38 *

Group 2:

167 0.36 3.86

232 5.96 3.97

233 1.54 1.91

543 0.52 0.00

381 3.90 3.83

549 2.23 2.20

2.42 # 2.63 #

* Overall means for group one. Infected mammary quarters

produced significantly less milk than the contralateral control

mammary quarters (P = 0.007).

# Overal means for group two. Milk production was not

significantly different between infected and control mammary

quarters (P = 0.834).

Group 1 = Novel strain infected cattle

Group 2 = Other strains of S. aureus

Cow 167 = ATCC 29740Cows 233 & 543 = Strain 233Cows 381 and 549 = Strain 381

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Biosecurity

• Study of 15 dairy herds in the PNW– The majority of strains of S. aureus that cause bovine

mastitis were unique to one herd.

– Herds that purchased replacement heifers had a higher prevalence of S. aureus mastitis than herds that purchased lactating cattle for herd expansion.

– Herds that purchased replacement heifers had more strains of S. aureus than closed herds.

– Herds that purchased replacement heifers acquired more new strains of S. aureus than closed herds.

S. aureus Genotype B

Term used by Swiss research group -> PCR amplification of 16S-23S rRNA intergenic spacer region (RS)-PCR

Identified 17 genotypes among 101 epidemiologically independent S. aureus isolates.

TWO genotypes (B and C) most common – 80.2% (81/101)

(Fournier et al, 2008; Graber et al, 2009)

Genotype B - associated with sed (84%), sej (80%), sea (77%), lukE (100%); highly contagious and considered pathogenic.

S. aureus Genotype B

(Fournier et al, 2008; Graber et al, 2009)

U.S. Isolates & Genotype B?

• 65 S. aureus isolates from 8 dairy farms in the Pacific Northwest

• 35 different PFGE strain-types

• NAGase and SCC data associated with each isolate

• RS-PCR (Jensen et al., 1993)

• Standard PCR was used to confirm the presence or absence of the leukotoxin E (lukE) gene and sea, sed, seg, sei, and sej.

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U.S. IsolatesGenotype (n=65) sea sed sei sej seg lukE

Y (n=1) 1 (100%) 0 0 0 0 1 (100%)

E (n=3) 0 0 3 (100%) 0 3 (100%) 3 (100%)

H (n=1) 0 0 1 (100%) 0 0 1 (100%)

I (n=43) 3 (6.9%) 0 2 (4.6%) 0 0 42 (98%)

X (n=1) 0 0 0 0 0 1 (100%)

Non-typable (n=16) 1 (6.3%) 0 2 (13%) 0 0 9 (56%)

Total (n=65) 5 (7.7%) 0 8 (12.3%) 0 3 (4.6%) 57 (87.7%)

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CONCLUSIONS• 35 PFGE strains versus 6 RS-PCR genotypes• No Genotype B found• No relationship between genotype, SCC, and NAGase

activity (P ≥ 0.64)• Discriminatory power of RS-PCR = 0.46 relative to

PFGE (1.0).

Detection & Diagnosis

• SCC/CMT– Sensitivity ranges from 0.86-0.96

• Definitive diagnosis based on detection of organism– Bacterial culture– PCR detection

• Source of sample; aseptic collection

– Strain-typing

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Treatment

• Probability of cure depends on cow, pathogen, and treatment factors (Barkema et al., 2006)– Cure rates: 3-74%

• Major factors impacting cure rate include:– Cow age

– Increasing SCC

– Chronicity

– Increasing bacteria count

– Increasing numbers of mammary quarters infected

– Duration of therapy

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S. Nickerson

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Treatment

• “Treatment of young animals with penicillin-sensitive S. aureus infections is often justified based on bacteriological cure and economic outcome, whereas treatment of older animals, chronic infections, or penicillin-resistant isolates should be discouraged.” (Barkema et al., 2006)

• Systematic Review of Literature (Roy & Keefe, 2012)

– Extended IMM therapy for 5-8 days was the best option

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Caution!

• Extended therapy can be associated with clinical mastitis with secondary organisms such as yeasts and coliforms (Roy & Keefe, 2012; Middleton & Luby, 2008)

• May constitute Extra-label Drug Use!

• Overall, long term cure rates even with 8 days of therapy can be quite poor (Middleton et al., 2007)

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Pre-partum Treatment of Heifers

• Caution! – Extra-label drug use.

• Cure rates can be quite high, but overall impact on 1st lactation performance over no treatment varies from herd-to-herd with no benefit in some herds.

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Prevention & Control

• Properly functioning milking equipment

• Gloves*

• Decrease potential for transmission

– Milking time hygiene

– Milk order

• Post-milking teat dip

• Dry cow therapy

• Cull chronics

• Avoid purchase of older cattle

• Pre-purchase screening (Biosecurity)

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Prevention & Control

• Outbreaks may occur in herds with excellent milking time hygiene (Smith et al., 1998).– May require more aggressive control program (Middleton

et al., 2001; Zeconni et al., 2003)• Segregation & milk last

• Dry-off infected quarters

• Accelerated culling

• In contrast, some herds have sporadic strains that are not highly transmissible (non-host-adapted S. aureus) and may not require intervention.

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Vaccination

A S. aureus mastitis vaccine should reliably

prevent intramammary infection because

the reservoir of infection is the cow’s udder

and the majority of transmission occurs

from cow-to-cow at the time of milking.

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Staphylococcus aureus Vaccines

• No vaccines studied to date consistently prevent new S. aureusIMI.

• Two commercial vaccines (Lysigin®, BIVI; Startvac®, HIPRA)

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Lysigin®

• Lysed polyvalent S. aureus bacterin

– 5 strains; 3 capsular serotypes (Ma et al., 2003)

• Serotype 5 – 1 strain

• Serotype 8 – 2 strains

• Serotype 336 – 2 strains

– Capsular serotypes 5, 8, and 336 are the most common mastitis causing S. aureus serotypes (Guidry et al., 1997 & 1999)

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Lysigin® in Heifers

• MU Heifer Challenge Trial– No difference in S. aureus IMI clearance (P ≥ 0.214)

– No difference in milk SCC (P ≥ 0.148)

– No difference in milk yield (P = 0.617)

– Cattle vaccinated with commercial Lysigin® had a lower mean duration of clinical mastitis and lower cumulative mastitis score than control (P = 0.045 & 0.046, respectively)

– No significant difference in milk opsonizing Ab (P > 0.05)

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Field Trial Data

• Lactating dairy cattle (44 vaccinates and 46 controls). – New S. aureus IMI

• 7/44 vaccinates and 4/46 controls (P = 0.511)

– New CNS IMI

• 18 of 157 at risk quarters in vaccinates– Incidence = 0.82 new IMI/quarter month

• 20 of 159 at risk quarters in controls– Incidence = 0.81 new IMI/quarter month

• Median time to new IMI = 56 days (P = 0.944)

– No significant difference in milk Ab (P > 0.30)

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Nickerson et al., 1999

• Early vaccination of heifers @ 6-months with 6-monthly follow-up until calving.– Staphylococcus aureus IMI

• 44.8% reduction in new IMI during pregnancy

• 43.1% reduction in IMIs becoming chronic

• 44.7% reduction in new IMI at freshening

– Other Staphylococcal spp.• 0.0% reduction in new IMI during pregnancy

• 34.7% reduction in IMI becoming chronic

• 30.8% reduction in new IMI at freshening

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Vaccination as an Adjunct to Treatment

• Adjunct to extended pirlimycin IMM therapy

• -14d, -7d, 2d relative to treatment

• Luby et al. 2005. Vet Record. 157, 89-90.

• Middleton et al. 2007. Proc Natl Mast Counc. 45, 232-233.

• No significant difference between vaccination and treatment versus treatment alone.

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Startvac®

• Multivalent mastitis vaccine

– Inactivated E. coli (J5 strain)

– Inactivated S. aureus (CP8) SP140 strain expressing slime associated antigenic complex (SAAC)

• Label – reduce incidence of subclinical mastitis and decrease the clinical severity of mastitis caused by S. aureus, coliforms, and CNS.

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Schukken et al., 2014

• 45% decrease in basic reproduction ratio for S. aureus

• 35% decrease in basic reproduction ratio for CNS

• Efficacy age dependent (> reduction in Parity 1 vs. 3 or >)

• Results varied between herds

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Schukken et al., 2014

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Startvac®

• Stimulates humoral immune response.

• Ameliorates inflammation in the udder following challenge with inactivated S. aureus.

• Does not completely prevent infection, but recent data suggests it may decrease S. aureus transmission rates within herd by reducing incidence and duration of IMI.

40Piepers et al., 2012; Schukken et al., 2014

Take Home Messages

• Have we learned anything about S. aureus mastitis in the last 50 years? YES!

• Has what we have learned changed the basic premise that milking time hygiene is the main critical control point? NO!

• Ultimately, decisions on how to manage S. aureus mastitis in a given herd will depend on the contagiousness, persistence, and inflammatory nature of the infecting strains.

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Take Home Messages

• Use of historical data on new IMI rates, SCC, and response to therapy will be valuable tools in making management decisions.

• Culture and strain-typing may be necessary in some herds.– If most infections are caused by a single strain that is refractory to

treatment, then culling may be the only option.– If strains are diverse and sporadic, drastic measures may not be needed.

• Vaccination against S. aureus will not completely prevent IMI, but some strategies may be beneficial in reducing transmission when used in conjunction with contagious mastitis pathogen control procedures outlined in the NMC 10-point plan.

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Questions?

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